KR19990007901A - Recombinant zodiac lipase and polypeptide derivatives produced by plants, methods for their preparation and uses thereof - Google Patents

Abstract

The present invention relates to the use for transforming plant cells to obtain a recombinant nucleotide sequence comprising a duodenal lipase, or a recombinant ileum lipase or polypeptide derivative of a sequence derived from this cDNA.

The present invention also relates to the use of a genetically modified plant or part thereof, or a plant extract thereof, or a recombinant plenum lipase or produced polypeptide derivative in food or pharmaceutical manufacturing or industry.

Description

Recombinant zodiac lipase and polypeptide derivatives produced by plants, methods for their preparation and uses thereof

The present invention relates to recombinant lipases by plants, in particular recombinant gastrointestinal lipases and their other polypeptide derivatives having lipase activity and their use, in particular functional food or pharmaceutical compositions or enzyme preparations for fertilizer or industrial applications.

Dog gastrointestinal lipase (DGL) is a glycoprotein of 379 amino acids of molecular weight about 50 kilodaltons (kDa) that has a signal peptide at its amino terminus (NH ₂ terminus) and is secreted by the central cells of the basal mucosa of the dog's stomach (Carriere F. et al., 1991).

Human gastrointestinal lipase (HGL) is naturally synthesized in the form of precursors and described in Bodmer et al., 1987. Mature HGL protein consists of 379 amino acids. Its signal peptide (HGLSP) consists of 19 amino acids.

These enzymes belong to a group of lipases called preduodenals, some of which are already purified and in some cases cloned (Docherty AJP et al., 1985; Bodmer MW et al., 1987; Moreau H. et al. , 1988; European Patent Nos. 0 191 061 and 0 261 016).

For a long time, hydrolysis of lipids in food was thought to occur in the small intestine by the action of enzymes produced in the pancreas.

However, it has been suggested that the hydrolysis of triglycerides can occur in the gastrointestinal tract by the indirect action of the twentiodenum enzymes (Volhard, F., 1901; Shonheyder, F., and Volquartz, K., 1945). These enzymes, particularly dog gastric lipases, have enzymatic and physicochemical properties that distinguish these enzymes from mammalian pancreatic lipases. These differences between gastrointestinal and pancreatic lipases are inherently related to molecular weight, amino acid composition, pepsin resistance, substrate specificity, optimal working pH and stability in acid medium.

It is also possible to demonstrate synergy between gastrointestinal and pancreatic lipases on the hydrolysis of long chain triglycerides in vitro under certain conditions (Gargouri, Y. et al., 1989).

Several pathological conditions (cystic fibrosis, pancreatic exocrine deficiency) have been known, in which the patient is wholly or partially lacking pancreatic exocrine secretion and lacks enzymes (amylase, lipase, protease) necessary for hydrolysis of food. Absorption of fat, especially triglycerides, in the intestine is evident by a very significant increase in fat diarrhea in these patients and a very significant decrease in weight gain in young patients. To correct this, the pig pancreatic extract is administered to the subject at mealtime. The therapeutic efficacy of the extract can be greatly improved by simultaneous prescription of DGL by the specificity of the action of DGL on long chain triglycerides.

Carriere et al. (1991) describe the purification of DGL and determination of the NH ₂ terminal sequence. Methods of extracting such enzymes from the stomach of dogs are also described in this document. The method consists essentially of dipping the stomach of the dog in the acid medium in the presence of a water soluble salt that promotes the salting out of the lipase in acid medium (pH 2.5). DGL can be homogeneously purified by the step of filtration on molecular sieves and ion exchange chromatography. Purified DGL obtained by the above method is a glycoprotein of 49,000 Daltons of molecular weight of 6,000 Daltons corresponding to sugar and 43,000 corresponding to protein portion.

It is difficult to develop the method in the laboratory or industrially because the acquisition of the dog's stomach is difficult. Because of this, there is a need to find a way to generate large amounts of DGL without having to use a dog's stomach.

Nucleotide and peptide sequences of DGL were determined with the aid of industrial production of DGL by methods using genetic engineering. This study was the subject of International Application Publication No. 94/131816, filed December 16, 1993.

The production method of recombinant DGL described in the above international application used Escherichia coli (E. coli) as a transformed host cell capable of producing DGL.

this. Some problems encountered during the production of recombinant DGL by E. coli, especially at high cost in fermenters. Because of the need to cultivate E. coli, the researchers studied other methods of producing the DGL.

Mammalian cells are more suitable for expression of mammalian genes. However, the use of these cells presents a problem of protein maturation. Enzymatic devices that achieve post-translational maturity differ from one tissue, organ or species to another tissue, organ, species. For example, post-translational maturation of plasma proteins may differ depending on whether they are obtained from human blood, generated from recombinant cells, eg, ovarian cells of Chinese hamsters, or present in the milk of transgenic animals. have. In addition, low expression levels obtained using mammalian cells entail culturing at high cost in vitro at very large volumes. The production of recombinant proteins in the milk of transgenic animals (mouse, sheep and cattle) makes it possible to reduce production costs and overcome the problem of expression levels. However, ethical issues and problems of viral and subviral contamination (prions) still exist.

For this reason, transgenesis of mammalian genes into plant cells can provide a route for mass production of new recombinant proteins at reduced production costs without the risk of viral or subviral contamination.

In 1983 several laboratories introduced heterologous genes into the genome of plant cells (Bevan et al., 1983; Herrera-Estrella et al., 1983 a and b) to generate transgenic plants from these genetically modified cells. I found it possible. Thus, all cells of the plant have genetically modified properties that are delivered to descendants by oily fertilization.

As a result of this study, many researchers have been interested in the production of mammalian recombinant proteins in plant cells or transgenic plants (Barta et al., 1986; Marx, 1982). One of the very important first results in this field was the production of antibodies in transgenic tobacco (Hiatt et al., 1989).

To express the heterologous protein in the seed, the plant's protein storage site, Vandekererckhove's team fused the leu-enkephalin coding sequence to the 2S albumin coding gene from Arabidopsis thaliana. This construct was used to make transgenic flats that specifically expressed leu-enkephalin at an expression level of 0.1% of the total protein in the seed. In 1990, Simon and his colleagues introduced human serum albumin genes into tobacco and potato cells. Regardless of the origin (human or plant) of the signal peptide, human serum albumin was obtained in potato leaves, stems and tubers with an expression level of 0.02% of the total protein.

In addition, other mammalian recombinant proteins have been produced in plants: surface antigens of hepatitis B (Mason et al., 1992), interferons (De Zoeten et al., 1989; Edelbaum et al., 1992; Truve et al., 1993); Murine anti-Streptococcus mutans antigen, caries material (Hiatt and Ma, 1992; Ma et al., 1994), scFV anti-cancer cell antibody (Russel D., 1994), anti-herpes antibody (Russel D., 1994) , Hirudin (Moloney et al. 1994), cholera toxin (Hein R. 1994) and human epidermal growth factor (EGF) (Higo et al., 1993).

All of these studies demonstrated that mammalian recombinant protein production in plant cells is possible and that the mechanism of protein synthesis from DNA sequences is similar in animal cells and plant cells. However, there are some differences between plant and animal cells, particularly the maturation of polymannoside glycans to glycan complexes or differences in the site of degradation of signal peptides, thereby activating plant cells to express active or sufficiently active mammalian proteins. It cannot be guaranteed that it can be obtained by conversion.

We have demonstrated that plant cells transformed with appropriate recombinant nucleotide sequences can produce recombinant DGL or recombinant HGL, or polypeptides derived from DGL or HGL with sufficient enzymatic activity on an industrial scale.

It is an object of the present invention to provide novel novel methods of recombinant zodiac lipase, specifically recombinant DGL or HGL, or polypeptides derived from DGL or HGL having sufficient enzymatic activity, specifically lipase activity, which can be used industrially in mammals by plants. To provide a method of generation.

Another object of the present invention is to provide a means for carrying out the method, in particular a novel recombinant nucleotide sequence, a genetically transformed plant cell, a genetically transformed organism or part of a plant (leaves, stems, fruits, seeds or grains, roots). ) And genetically transformed fragments of these plants or parts thereof.

It is also an object of the present invention to provide novel mammalian recombinant pleural lipase (s), or inducible polypeptides obtained from genetically transformed plant cells or plants with enzymatic activity.

Another object of the present invention is to provide new enzyme compositions that can be used to carry out enzymatic reactions, particularly on an industrial scale.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of lesions, in particular related to the lack of lipase production in organisms such as cystic fibrosis.

Another object of the present invention is to provide a new fuel, so-called biofuel, which has the advantage of less pollution and lower production cost than fuel derived from petroleum.

The present invention will be described with reference to the following drawings.

FIG. 1 shows the nucleotide sequence of the cDNA encoding the DGL shown in FIG. 2 in which the nucleotides 1 to 1,137 in the sequence correspond to SEQ ID NO: 2.

2 shows the amino acid sequence of DGL corresponding to SEQ ID NO: 2.

FIG. 3 shows the nucleotide sequence corresponding to SEQ ID NO: 1 derived from the cDNA shown in FIG. 1, wherein the nucleotides present in 1 to 1,137 of the derived sequence encode the DGL shown in FIG. do.

4 shows the nucleotide sequence of a cDNA in which the nucleotides at 47-1240 in the sequence encode the precursor of HGL of 398 amino acids and the nucleotides at 104-1240 encode the mature HGL of 379 amino acids.

5 shows the amino acid sequence of HGL, where the precursor of HGL consists of amino acids present in 1 to 398 and the mature HGL consists of amino acids present in 20 to 398.

6, 7 and 8 show western-type immunodetection of recombinant polypeptides produced by leaves and seeds, rapeseed and tomato leaves and fruits of tobacco Xanthi transformed with the recombinant sequence according to the present invention. The experimental results are shown.

9 and 10 show the results of analysis by electrophoresis on polyacrylamide gels and the display of the gels from dog gastrointestinal anti-lipase antibodies prior to transfer onto nitrocellulose membranes and purified from tobacco leaves. will be.

11 shows an example of detection on the membrane of glycan residues of recombinant DGL.

FIG. 12 shows the analysis by electrophoresis on polyacrylamide gel of DGL purified in rapeseed.

13, 14 and 15 illustrate various tests conducted in the preparation of methyl oleate from seed transformed according to the present invention.

The present invention, on the one hand, has a homology of at least about 75%, in particular from about 77% to 85%, and has an amino acid sequence of at least 70%, in particular from about 80% to about nucleotide, encoding any mammalian ileum lipase CDNA encoding any mammalian ileum lipase having a homology of 90% and having properties that are acid resistant and active at a pH of about 1 to about 5, especially about 1.5 to about 2, more specifically any mammal Any polypeptide derived from said duodenal lipase by addition and / or inhibition and / or substitution of one (or more) amino acid (s) having the above properties of an animal gastrointestinal lipase or cDNA encoding CDNA encoding, on the other hand, to allow plant cells to produce pleural lipases encoded by the cDNA or to produce induced polypeptides as defined above Mammalian recombinant transduodenal lipase in the form of an active enzyme from a transformed plant cell or a plant obtained from the cell, the recombinant nucleotide sequence comprising a transcriptional promoter and a transcription terminator recognized by the transcriptional machinery of the plant cell, or It relates to the use for transforming plant cells to obtain one (or more) polypeptide (s) defined.

More specifically, the invention on the one hand adds and / or inhibits the cDNA, or one (or more) nucleotide (s), shown in FIG. 1 encoding dog gastrointestinal lipase (DGL), as shown in FIG. Or) addition of a nucleotide sequence capable of encoding a polypeptide of amino acid sequence identical to the amino acid sequence of DGL shown in FIG. 2 derived from the cDNA by substitution, or one (or more) amino acid (s), and / or Nucleotides encoding polypeptides having lipase activity, derived from DGL by inhibition and / or substitution, on the other hand, components which allow plant cells to produce polypeptides encoded by the cDNA or the derived sequences, in particular plants Recombinant nucleophiles comprising a transcriptional promoter and transcriptional terminator recognized by the transcriptional machinery of the cell (more specifically RNA polymerase of a plant cell) De-sequence, for use in transforming plant cells to obtain recombinant DGL in active enzyme form or one (or more) derived polypeptide (s) as defined above from a transformed plant cell or a plant obtained from the cell. It is about.

The invention also relates to the recombinant nucleotide sequence comprising the cDNA shown in FIG. 1 or the derived nucleotide sequence as defined above.

In this aspect, the present invention more specifically relates to the recombinant nucleotide sequence comprising the cDNA shown in FIG. 1 encoding dog gastrointestinal lipase (DGL) shown in FIG. 2.

The present invention further relates to the above recombinant nucleotide sequence comprising the nucleotide sequence derived from cDNA shown in Fig. 1 as defined above and encoding the dog gastrointestinal lipase (DGL) shown in Fig. 2. The derived sequence is advantageous in the sequence shown in FIG. 3, wherein in the sequence shown in FIG. 1, nucleotide A at position 12 is replaced by nucleotide G, nucleotide T at position 13 is replaced by nucleotide C, and nucleotide at position 15 is nucleotide. Corresponds to the sequence substituted with T.

Recombinant nucleotide sequences according to the present invention allow the recombinant polypeptide (i.e., DGL or the derived polypeptide) of the present invention to be incorporated into specific compartments of plant cells, in particular vesicles or vacuoles, or outside the cells, into the pectulocellulose wall or extracellular space. It is advantageous to include one (or more) sequence (s) encoding a peptide which functions to direct the so-called apoplast.

Transcription terminators that can be used for transformation of plant cells in the present invention include terminator polyA 35S of Cauliflower Mosaic Virus (CaMV) described in Franck et al., 1980 or Agrobacterium tumefaciens with nopalin. Reference may be made to the polyA NOS terminator (Depicker et al., 1982) corresponding to the 3 'noncoding region of the gene of the nopalin synthase of plasmid Ti of the Agrobacterium tumefaciens strain.

In this aspect the invention relates to said recombinant nucleotide sequence comprising terminator polyA 35S of CaMV or terminator polyA NOS of Agrobacterium tumefaciens downstream of said cDNA or its derived sequence.

In the present invention, as a transcriptional promoter that can be used for transformation of plant cells, mention may be made of the following promoters:

Promoter 35S or advantageously double-stranded of CaMV as described in Kay et al., 1987, which enables expression of recombinant polypeptides according to the invention in whole plants obtained from cells transformed according to the invention. Promoter 35S (pd35S),

-Radish cruciferin described in Depigny-This et al., 1992, which enables expression of the recombinant polypeptides of the invention only in the seed (or kernel) of the plant obtained from the cells transformed according to the invention. Promoter of gene pCRU,

Promoters pGEA1 and pGEA6, corresponding to the 5 'uncoding regions of the genes of the genes of GEA1 and GEA6, the storage proteins of the grains of Arabidopsis thaliana, respectively (Gaubier et al., 1993) and allowing specific expression in the grains,

By fusion of the three transcriptional activators of the promoter of the octopin gene of Agrobacterium tumefaciens, the transcriptional activator component of the promoter of the mannopin synthase of Agrobacterium tumefaciens, and the promoter of the mannopin synthase The resulting chimera promoter, super-promoter pSP (PCT / US94 / 12946),

To the actin promoter of rice present after the actin intron of rice contained in the plasmid pAct1-F4 described in McElroy et al., 1991 (pAR-IAR),

A promoter of the maize γzeine gene described in Reina et al., 1990 and contained in the plasmid pγ63 that allows expression of maize seed in the embryo.

In view of the above, the present invention relates to a double-stranded promoter 35S (pd35S) of CaMV or a promoter pCRU or a promoter of Arabidopsis thaliana pGEA1 or pGEA6 or Agrobacterium tumefaciens of CaMV And to said recombinant nucleotide sequence comprising a super-promoter pSP or rice promoter pAR-IAR or maize promoter pγzeine.

The sequence encoding the directed peptide for use in the present invention may be of plant, human or animal origin.

Sequences encoding directed peptides of plant origin include:

A nucleotide sequence of 69 nucleotides encoding the 23 amino acid prepeptide (signal peptide) of sporamin A of sweet potato, which introduces the recombinant polypeptide of the invention into the secretory (mainly endoplasmic reticulum) of the transformed plant cell according to the invention (Shown in the Examples below),

A nucleotide sequence of 42 nucleotides encoding the N-terminal vacuole directed peptide of 14 amino acids of sporamin A of sweet potato, which accumulates in the vacuoles of the transformed plant cells according to the invention (Examples below) ),

A free consisting of 23 amino acids of the C terminus at the N terminus of the signal peptide, followed by 14 amino acids of the propeptide, in which the recombinant polypeptide of the present invention is introduced into the secretory system of the transformed plant cell according to the present invention and accumulates in the vacuole Nucleotide sequence of 111 nucleotides encoding the propeptide (shown in the Examples below), wherein the three sequences are described in Murakami et al., 1986 and Matsuoka and Nakamura, 1991)

The carboxy terminal propeptide of the lectins of barley as described in Schreder et al., 1993 and Bednarek and Ralkhel, 1991.

Sequences encoding directional peptides of human or animal origin include signal peptides of human gastrointestinal lipase (HGL) described in European Patent No. 0 191 061 or rabbit gastrointestinal lipase described in European Patent Application No. 0 542 629 shown in the Examples below. Mention may be made of a signal sequence of (RGL) or a signal sequence of human pancreatic lipase (HPL) or a sequence encoding a signal sequence of dog gastrointestinal lipase (DGL).

As the sequence encoding the directed peptide, a sequence encoding KDEL, which is a tetrapeptide directed to the endoplasmic reticulum, may be mentioned.

In this aspect the invention relates to said recombinant nucleotide sequence comprising a sequence encoding all or part of a signal peptide such as a signal peptide of sporamin A of sweet potato or a signal peptide of HGL or RGL or DGL, encoding a signal peptide Wherein said sequence is present upstream of said cDNA or derived sequence thereof within said recombinant nucleotide sequence and downstream of the promoter used so that the final C-terminal amino acid of the signal peptide in the protein encoded by said recombinant nucleotide sequence is said cDNA Or a first N-terminal amino acid of a polypeptide encoded by its derived sequence.

The invention also relates to the recombinant nucleotide sequence comprising a sequence encoding all or a portion of a vacuole-directed peptide, in particular the vacuole-directed peptide of sporamin A in sweet potato, wherein the sequence encoding the vacuole-directed peptide is the recombinant nucleotide. The first N-terminal amino acid of the vacuole directed peptide in the protein encoded by the recombinant nucleotide sequence between the sequence encoding the signal peptide and the sequence encoding the cDNA or its derived sequence within the sequence is the final Is bound to the C-terminal amino acid, and the final C-terminal amino acid of the directed peptide is bound to the first N-terminal amino acid of the polypeptide encoded by the cDNA or its derived sequence.

The invention also relates to said recombinant nucleotide sequence comprising a sequence encoding all or a portion of a vacuole-directed peptide, in particular the vacuole-directed peptide of lectins of barley, wherein said sequence encoding a vacuole-directed peptide is in said recombinant nucleotide sequence. A polypeptide that is downstream of a sequence encoding the cDNA or its derived sequence, wherein the first N-terminal amino acid of the vacuole directed peptide in the protein encoded by the recombinant nucleotide sequence is encoded by the cDNA or its derived sequence Is bound to the final C-terminal amino acid.

More specifically, the present invention relates to the following recombinant nucleotide sequences:

A nucleotide sequence comprising a sequence encoding the promoter pd35S of CaMV, the signal peptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV in the 5 '→ 3' direction ( pd35S-PS-DGL),

A nucleotide sequence comprising a sequence encoding the promoter pd35S of CaMV, the prepropeptide of sporamin A in the 5 '→ 3' direction, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV (called pd35S-PPS-DGL),

The sequence encoding the promoter pd35S of CaMV, part of the signal peptide of RGL in the 5 '→ 3' direction (with the first 19 amino acids suppressed with 9 nucleotides encoding the last three C-terminal amino acids shown in the Examples below) Constructed sequence), the nucleotide sequence (called pd35S-RGLSP-DGL), which in turn comprises the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 immediately after this sequence,

A nucleotide sequence comprising a promoter pCRU of cruciferin in the 5 '→ 3' direction, a sequence encoding a signal peptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and a terminator polyA 35S of CaMV (called pCRU-PS-DGL),

A nucleotide comprising the promoter pCRU of cruciferin in the 5 '→ 3' direction, the sequence encoding the prepropeptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV. Sequence (called pCRU-PPS-DGL),

-A sequence encoding a part of the signal peptide of the cruciferin promoter pCRU, RGL in the 5 '→ 3' direction (same as above), and the terminator polyA 35S of the cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. Nucleotide sequence comprising in sequence (called pCRU-RGLSP-DGL),

-A sequence encoding the part of the signal peptide of the Arabidopsis thaliana promoter pGEA1, RGL in the 5 '→ 3' direction (same as above), the terminator polyA 35S of the cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. A nucleotide sequence comprising in sequence (called pGEA1-RGLSP-DGL),

-A sequence encoding a part of the signal peptide of RAVIDOPDIS THALIANA promoter pGEA6, RGL in the 5 '→ 3' direction (same as above), the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 or 3 immediately after this sequence A nucleotide sequence comprising in sequence (called pGEA6-RGLSP-DGL),

A sequence encoding a part of the rice promoter pAR-IAR, a signal peptide of RGL in the 5 '→ 3' direction (same as above), and the terminator polyA of cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. A nucleotide sequence comprising the terminator polyA NOS of 35S or Agrobacterium tumefaciens (called pAR-IAR-RGLSP-DGL),

The sequence encoding the promoter pγzeine of corn, part of the signal peptide of RGL in the 5 '→ 3' direction (same as above), and the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 or 3 immediately after this sequence. Nucleotide sequence comprising in sequence (called pγzeine-RGLSP-DGL),

A sequence encoding the promoter pγzeine of corn and a part of the signal peptide of RGL in the 5 '→ 3' direction (same as above), the sequence encoding the cDNA and tetrapeptide KDEL shown in FIG. 1 or 3 immediately after the sequence. And a nucleotide sequence comprising the terminator polyA 35S of CaMV in order (called pγzeine-RGLSP-DGL-KDEL),

In addition, the recombinant nucleotide sequence of the present invention is particularly advantageous to include a nucleotide sequence which can be used as a marker of the recombinant sequence for distinguishing (and screening) plant cells transformed with the recombinant sequence and untransformed cells. Do.

The nucleotide sequence that can be used as a marker of the recombinant sequence is preferably selected from genes that are resistant to antibiotics, particularly kanamycin.

The invention also relates to a vector, in particular a plasmid vector, comprising the recombinant nucleotide sequence according to the invention inserted at a site which is not essential for its replication.

The invention also relates to host cells, in particular bacteria, for example Agrobacterium tumefaciens, transformed with a vector as defined above.

In addition, the present invention

Transforming the plant cell such that one (or more) recombinant nucleotide sequence (s) according to the invention is integrated into the genome of the plant cell,

If appropriate, generating a transformed plant from said transformed cells,

Lipase activity derived from said recombinant DGL by addition and / or inhibition and / or substitution of recombinant DGL and / or in particular one (or more) amino acid (s) produced in said cell or said transformed plant. Recovering the polypeptide (s) having a specific viability by extraction, and, where appropriate, by purification after extraction

Reactive DGL in the form of an active enzyme and / or a method for producing a polypeptide (s) having lipase activity derived from recombinant DGL by the above method, characterized in that consisting of.

According to one embodiment of the invention, the transformation of plant cells is carried out by the recombinant nucleotides of the invention, in particular the protoplasts in the presence of a divalent cation (Ca ²⁺ ) according to the method described in Krens et al., 1982. It can be done by incubating in glycol (PEG) solution and then introducing it into the plasma.

In addition, transformation of plant cells can be carried out by electroporation, in particular according to the method of Fromm et al., 1986.

Transformation of plant cells can also be carried out using a gene gun which delivers genes into the cell nucleus by expressway firing metal particles coated with the recombinant nucleotide sequence according to the invention by the technique described in Sanford, 1988.

Another method for carrying out transformation of plant cells is the cytoplasmic or nuclear microinjection method described in De La Penna et al., 1987.

According to a particularly preferred embodiment of the method of the present invention, the plant cell is transformed with the vector according to the present invention and infects the plant cell so that the nucleotide sequence of the present invention initially contained in the genome of the vector is used for the plant cell. The cell host is transformed by positioning it with the plant cell, which can integrate into the genome.

The cell host used is in particular Agrobacterium tumefaciens according to the method described in Bevan, 1984 and An et al., 1986 or Agrobacterium risico according to the method described in Joinin et al., 1987. Gennes (Agrobacterium rhizogenes).

Plant cells that can be transformed in the present invention may include rape, tobacco, corn, peas, tomatoes, carrots, wheat, barley, potatoes, soybeans, sunflowers, lettuce, rice and alfalfa.

According to one embodiment of the method of the present invention, the transformed plant cell according to the present invention is in accordance with the method described in Brodelius, 1988, or in liquid medium, or according to the method of Brodelius et al., 1979. It is cultured in fixed form in vitro, in particular in a bioreactor or by incubation of transformed roots in vitro according to the methods of Deno et al., 1987.

Subsequently, the recombinant DGL produced by the transformed cells cultured in vitro and / or the defined polypeptide (s) defined above are extracted from the in vitro culture medium and purified by chromatography if appropriate. It is recovered.

According to a preferred embodiment of the above method for producing recombinant DGL and / or derived polypeptide (s) according to the present invention, transformed plants are produced by culturing the transformed cells in a suitable medium after transformation of the plant cells. To perform the step. The recombinant DGL and / or derived polypeptide (s) produced in the cells of the whole plant thus obtained are carried out on the whole plant or part of the plant (especially leaves, stems or fruits), or seeds produced by the plant. Extraction is followed by extraction, if appropriate, for purification of the recombinant DGL and / or derived polypeptide (s).

The transformed plants used for the recovery of recombinant DGL and / or derived polypeptide (s) in the method are those of generation T0, advantageously obtained by culturing the transformed cells of the invention in a suitable medium. Is a plant of the next generation (T1, T2, etc.) obtained by self-correction of the plant of the previous generation and the recombinant nucleotide sequence of the present invention is reproduced according to Mendel's law.

Polypeptides derived from recombinant DGL obtained by carrying out the methods of the present invention may include the following polypeptides:

The polypeptide shown in SEQ ID NO: 4 consisting of the amino acids at positions 55 to 379 of FIG. 2 and encoded by the nucleotide sequence shown in SEQ ID NO: 3 (Δ54),

The polypeptide shown in SEQ ID NO: 6 consisting of the amino acids at positions 5 to 379 in Figure 2 and encoded by the nucleotide sequence shown in SEQ ID NO: 5 (Δ4).

More specifically, the present invention relates to a method in which the transformation of plant cells comprises cDNA, on the one hand, as shown in FIG. 1 and on the other hand the signal peptide of 22 amino acids of RGL, advantageously the first 19 amino acids of the signal peptide of RGL. Production of the recombinant DGL shown in FIG. 2, and, if appropriate, one (or more) derived polypeptide (s), in particular characterized by the integration into said genome of said recombinant sequence comprising a sequence encoding To said method for the production of said polypeptide (Δ54) and / or polypeptide (Δ4).

More specifically, the present invention,

Transforming explant cells of the leaves of the plant by placing them on a suitable culture medium with a strain of Agrobacterium tumefaciens transformed by the plasmid comprising the recombinant nucleotide sequence pd35S-RGLSP-DGL Steps,

Selecting a transformed explant on a medium comprising kanamycin,

Production of the transformed plant from the explant by culturing the transformed explant on a suitable medium,

Recombinant DGL by recovery of the supernatant constituting the plant extract with milling, centrifugation and enzymatic activity of the leaves and / or seeds and / or fruits of said transformed plant in a suitable buffer, and, if appropriate, a polypeptide (Δ54). ) And / or extraction of the polypeptide (Δ4),

If appropriate, chromatography of the extract obtained during the above step, in particular supernatant, to purify the recombinant DGL to produce the recombinant DGL in essentially pure form.

A method for producing the recombinant DGL shown in FIG. 2 together with the polypeptide (Δ54) and / or polypeptide (Δ4), where appropriate.

In addition, the present invention provides a method of preparing said polypeptide (Δ54) and / or polypeptide (Δ4) in essentially pure form from the extract obtained in the above method, in particular by purification by chromatography on the supernatant of the extract. It is about a use.

More specifically, the present invention provides for the preparation of said polypeptide (Δ4) in essentially pure form if the cells transformed with the sequence pd35S-RGLSP-DGL are suitable by the practice of the above method, wherein the cell is an explant cell of an eggplant, in particular tobacco or tomato. It is about.

According to one embodiment of the method, the polypeptide (Δ4) can be specifically obtained by extraction from the tobacco leaf by grinding, centrifugation and recovery of the supernatant in the transformed tobacco leaf in a suitable buffer. The polypeptide (Δ4) can then be purified by chromatography on the supernatant from the leaf extract comprising the polypeptide (Δ4).

More specifically, in the present invention, the genome of the plant of the sequence in which the transforming step of the plant cell comprises on the one hand the nucleotide sequence shown in FIG. 3 and on the other hand the sequence encoding the signal sequence of the sporamin A It relates to the above method for the production of recombinant DGL and / or one or more (or more) polypeptide (s) as defined above, characterized in that it is carried out by integration into.

Polypeptides derived from recombinant polypeptides obtainable by the practice of the method may refer to such polypeptides (Δ54) and / or polypeptides (Δ4).

More specifically, the present invention,

Explants of plants (especially explants of leaves) by placing them together with strains of Agrobacterium tumefaciens transformed by said plasmids comprising recombinant nucleotide sequences pd35S-PS-DGL and / or pd35S-PPS-DGL ) Transforming the cells,

Selecting a transformed explant on a medium comprising kanamycin,

Production of the transformed plant from the explant by culturing the transformed explant on a suitable medium,

Recombinant DGL and / or polypeptide (Δ54) by recovery of the supernatant constituting the plant extract with milling, centrifugation and enzymatic activity of the leaves and / or seeds and / or fruits of said transformed plant in a suitable buffer. ) And / or extraction of the polypeptide (Δ4),

If appropriate, chromatography of the extracted DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) from the extract obtained during this step, in particular the supernatant, to purify the recombinant DGL and / or polypeptide ( Preparing Δ54) and / or polypeptide (Δ4) in essentially pure form

It relates to a method for producing the recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4).

More specifically, the present invention relates to a method for producing said polypeptide (Δ54) and / or polypeptide (Δ4) by the execution of said method wherein the transformed cells are eggplants, in particular explant cells of tobacco or tomato leaves.

According to one embodiment of the method, polypeptide (Δ54) can be obtained specifically by extraction from the tobacco seeds by grinding, centrifugation and recovery of the supernatant of the transformed tobacco seeds in a suitable buffer. The polypeptide (Δ54) can then be purified by chromatography on the supernatant from the seed extract comprising the polypeptide (Δ54).

More specifically, the present invention,

Recombinant nucleotide sequence pCRU-PPS-DGL and / or sequence pCRU-PS-DGL and / or sequence pGEA1-RGLSP-DGL and / or sequence pGEA6-RGLSP-DGL and / or sequence pAR-IAR-RGLSP- Explant cells of plants by placing them together with strains of Agrobacterium tumefaciens transformed by said plasmid comprising DGL and / or sequence pγzeine-RGLSP-DGL and / or sequence pγzeine-RGLSP-DGL-KDEL Transforming,

Selecting a transformed explant on a medium comprising kanamycin,

Production of the transformed plant from the explant by culturing the transformed explant on a suitable medium,

Of recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) by grinding, centrifugation and recovery of the supernatant constituting the plant extract with enzymatic activity of the seed of the transformed plant in a suitable buffer Extraction step,

If appropriate, chromatography of the extracted DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) from the extract obtained during this step, in particular the supernatant, to purify the recombinant DGL and / or polypeptide ( Preparing Δ54) and / or polypeptide (Δ4) in essentially pure form

It relates to a method for producing the recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4).

More specifically, the present invention relates to a method for producing the polypeptide (Δ54) by performing the above method wherein the transformed cells are explant cells of rapeseed and tobacco and the polypeptide (Δ54) is extracted by crushing the transformed seed. It is about.

The transformed plant cells in this method are advantageously selected from tobacco, rapeseed, corn, peas, tomatoes, carrots, wheat, barley, potatoes, soybeans, sunflowers, lettuce, rice and alfalfa.

More specifically, the object of the present invention

With a plasmid comprising the recombinant nucleotide sequence pAR-IAR-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL-KDEL by impacting the corn callus using a particle gun. Transforming the sewage callus,

Screening the transformed callus on a medium comprising a selection material such as kanamycin,

Production of maize transformed from the callus by culturing the transformed callus on a suitable medium,

Recombinant DGL and / or polypeptides (Δ54) and / or polypeptides (by crushing, centrifugation and recovery of supernatant constituting plant extracts having enzymatic activity of seeds produced by the transformed plants in a suitable buffer) Extraction step of Δ4),

If appropriate, chromatography of the extracted DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) from the extract obtained during this step, in particular the supernatant, to purify the recombinant DGL and / or polypeptide ( Preparing Δ54) and / or polypeptide (Δ4) in essentially pure form

It is characterized in that it comprises a recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4).

The invention also relates to a genetically transformed plant comprising said one (or more) recombinant nucleotide sequence (s) integrated in its genome in a stable manner according to the invention.

The invention also encompasses one (or more) recombinant polypeptide (s) according to the invention, such as recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) and includes enzymatic activity, in particular as defined below. The transgenic plant cell has lipase activity.

The invention also relates to a genetically transformed seed comprising said one (or more) recombinant nucleotide sequence (s) integrated in its genome in a stable manner according to the invention.

The invention also encompasses one (or more) recombinant polypeptide (s) according to the invention, such as recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) and includes enzymatic activity, in particular as defined below. It relates to said transgenic seed having lipase activity.

Seeds transformed according to the present invention are seed plants of said generation T0 produced by culturing the transformed cells according to the invention or progeny generation obtained by self fertilization or heterogeneous mating of the preceding generation (as described above) ( T1, T2, etc.) harvested from genetically transformed plants.

The invention furthermore comprises a genetically transformed plant or part of a plant, in particular, characterized in that it comprises said one (or more) recombinant nucleotide sequence (s) integrated in its genome in a stable manner according to the invention. Explants, stems, leaves, roots, pollen, etc.).

The invention also encompasses one (or more) recombinant polypeptide (s) according to the invention, such as recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) and includes enzymatic activity, in particular as defined below. A transgenic plant or part of a plant having lipase activity.

More specifically the present invention relates to said transformed plant obtained by culturing said cell or seed according to the present invention.

The plant, or part of the plant, transformed according to the present invention is selected from tobacco, rapeseed, corn, peas, tomatoes, carrots, wheat, barley, potatoes, soybeans, sunflowers, lettuce, rice and alfalfa or some of these plants. It is advantageous.

The invention comprises one (or more) recombinant polypeptide (s) according to the invention with enzymatic activity, in particular lipase activity as defined below, such as recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4). It relates to a plant extract prepared by carrying out the method of the present invention.

Lipase (or lipolytic) activity of plants or plant parts and plant extracts having the enzymatic activity of the present invention is described in Garouri et al., 1986, using short-chain triglycerides (eg tributyrin) as substrates. It can be measured according to the method. Enzyme activity is expressed in units (U) and one unit (U) corresponds to the amount of enzyme required to produce 1 μmol of free fatty acids per minute at 37 ° C. under optimal pH conditions.

Plant extracts having the enzymatic activity of the present invention may be from about 0.5 U to about 1,000 U / g (raw weight of the leaves (FM)), in particular from about 10 U / g (FW) to about 300 U / g (FW), or about The percent by weight of the recombinant polypeptide having enzymatic activity corresponds to a measurement of from 1 U to about 5,000 U / g (FM of seed), in particular from about 10 U / g (FW) to about 1,000 U / g (FW). It is advantageously from about 0.1% to 20%, in particular from about 1% to 15%, based on the total weight of the protein present in the.

More specifically, the present invention relates to the following plant extracts having enzymatic activity:

* Of explant cells of said plant using sequence pd35S-RGLSP-DGL or sequence pd35S-PS-DGL or sequence pd35S-RGLSP-DGL or sequence pd35S-PS-DGL or sequence pd35S-PPS-DGL according to one of the above methods Extracts of leaves and / or fruits and / or seeds of plants comprising recombinant DGL and / or polypeptides (Δ54) and / or polypeptides (Δ4), obtained by transformation, in particular

Containing the polypeptide (Δ54) together with the polypeptide (Δ4) as an extract of the tobacco leaf obtained by transformation of explant cells of the tobacco leaf using the sequence pd35S-PS-DGL or the sequence pd35S-PPS-DGL according to the above method And the weight percent of the mixture of polypeptide (Δ54) and polypeptide (Δ4) is from about 0.1% to about 20% based on the total weight of protein present in the extract and the enzyme activity of the extract is about 100 U / g (FW) Extract of tobacco leaves, which is from about 300 U / g (FW),

A polypeptide (Δ54) as an extract of a tomato leaf or fruit obtained by transformation of explant cells of tomato leaves using the sequence pd35S-PS-DGL or the sequence pd35S-PPS-DGL according to the above method. Together, the weight percent of the mixture of polypeptide (Δ54) and polypeptide (Δ4) is from about 0.1% to about 20% based on the total weight of protein present in the extract and the enzyme activity of the extract is about 100 U / g ( FW) to about 300 U / g (FW) extract of tomato leaves or fruits,

Extract of tobacco leaf obtained by transformation of explant cells of tobacco leaf using the sequence pd35S-RGLSP-DGL according to the above method, wherein the polypeptide (Δ4) is about 0.1 based on the total weight of the protein present in the extract Extract of tobacco leaves, comprising from about 20% by weight to about 20% by weight and having an enzyme activity of about 100 U / g (FW) to about 300 U / g (FW),

The total weight of protein present in the extract as polypeptide (Δ54) as an extract of tobacco seed obtained by transformation of explant cells of tobacco leaves using the sequence pd35S-PS-DGL or pd35S-PPS-DGL according to the above method An extract of tobacco leaves comprising from about 0.1% to about 1% by weight and having an enzyme activity of about 10 U / g (FW) to about 300 U / g (FW),

Recombinant DGL obtained by transformation of explant cells of a plant using the sequence pCRU-PS-DGL or the sequence pCRU-PPS-DGL or the sequence pGEA1-RGLSP-DGL or the sequence pGEA6-RGLSP-DGL according to the above method and (Or) extracts of plant seeds comprising polypeptides (Δ54) and / or polypeptides (Δ4), in particular

Extract of rapeseed obtained by transformation of explant cells of a plant using sequence pCRU-PS-DGL or sequence pCRU-PPS-DGL or sequence pGEA1-RGLSP-DGL or sequence pGEA6-RGLSP-DGL according to the above method And polypeptide (Δ54) from about 0.1% to about 1% by weight based on the total weight of proteins present in the extract and the enzyme activity of the extract is from about 10 U / g (FW) to about 1,000 U / g (FW Extract of rapeseed, which is

Obtained by transformation of explant cells of a plant using the sequence pAR-IAR-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL-KDEL according to the above method. Extracts of plant seeds comprising recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4), in particular

Obtained by transformation of explanted cells of rapeseed leaves using the sequence pAR-IAR-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL-KDEL according to the above method. As an extract of corn seed, the polypeptide (Δ54) comprises from about 0.1% to about 1% by weight based on the total weight of the protein present in the extract and the enzyme activity of the extract is from about 10 U / g (FW) to about 1,000 U Extract of Corn Seed, which is / g (FW).

In addition, the invention is shown in FIG. 2, which is obtained in essentially pure form by carrying out one of the methods of the invention comprising the step of purifying the recombinant polypeptide of the invention by chromatography performed on the enzyme extract. The present invention relates to a polypeptide having enzymatic activity of the amino acid sequence recombinant DGL, or lipase activity derived therefrom.

As a polypeptide derived from the recombinant DGL, the present invention relates more specifically to the polypeptides (Δ54) and (Δ4) having a molecular weight of about 37 kDa and about 49 kDa, respectively.

Enzymatically active recombinant DGL or derived polypeptide having lipase activity is understood to mean a recombinant polypeptide which may have lipase activity measured according to the method of Gargouri, supra.

For example, a recombinant polypeptide according to the present invention has a lipase activity of about 10 U / mg (recombinant polypeptide) to about 1,000 U / mg, advantageously about 100 U / mg to about 600 U / mg.

More specifically, the present invention is obtained by purifying the enzyme extract of tobacco leaves or seeds derived from transformed tobacco obtained from tobacco cells transformed with the sequence pd35S-RGLSP-DGL according to the above method. It relates to a recombinant DGL having activity.

The present invention also relates to transformed plants obtained from plant cells transformed with the sequence pd35S-PS-DGL or the sequence pd35S-PPS-DGL, or the sequence pd35S-RGLSP-DGL according to the above method, among which e. For example, the present invention relates to recombinant polypeptides (Δ54) and (Δ4) having lipase activity, which are obtained by purifying the enzyme extract of leaves and / or seeds and / or fruits of tobacco or tomatoes.

In addition, the present invention relates to tobacco seeds or rapeseed seeds derived from transformed tobacco or rapeseed, respectively, obtained from tobacco or rapeseed cells transformed with the sequence pCRU-PS-DGL or the sequence pCRU-PPS-DGL according to the above method. The present invention relates to a recombinant polypeptide having a lipase activity (Δ54), obtained by purification of an enzyme extract.

The present invention also provides for the purification of enzyme extracts of rapeseed seeds derived from transformed rapeseed obtained from rapeseed cells transformed with the sequence pGEA1-RGLSP-DGL and / or the sequence pGEA6-RGLSP-DGL according to the above method. The present invention relates to recombinant polypeptides (Δ54) and (Δ4) having the lipase activity obtained.

The present invention also provides a transformation obtained from corn cells transformed with the sequence pAR-IAR-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL and / or the sequence pγzeine-RGLSP-DGL-KDEL according to the above method. The present invention relates to recombinant polypeptides (Δ54) and (Δ4) having the lipase activity, which are obtained by purification of an enzyme extract of corn seeds derived from dried corn.

The apparent molecular weights of the recombinant polypeptides (Δ54) and (Δ4) according to the present invention were determined by analysis on polyacrylamide gels and immunodetection after electrophoresis on nitrocellulose, respectively (this method is described in the Examples of the Above) 37 kDa and 49 kDa.

The present invention is directed to antibodies directed against the recombinant polypeptides of the invention, more particularly to recombinant DGLs according to the invention and / or to said polypeptides (Δ54) and / or said polypeptides (Δ4), and also to recognize HGLs. It relates to an antibody that can be.

The antibody may be obtained by recovering an antibody formed after immunizing an animal with the polypeptide.

The product is of course not limited to polyclonal antibodies.

It can also be formed by conventional methods from spleen cells of animals, in particular mice or mice, which are immunized against one of the purified polypeptides of the invention on the one hand and from the suitable myeloma cells on the other hand, initially immunized. It applies to monoclonal antibodies produced by hybridomas that can be selected according to the polypeptides used in the present invention and the ability to produce monoclonal antibodies that recognize HGL.

The invention also relates to the invention for the preparation of one (or more) recombinant polypeptide (s) as defined above, such as DGL or a derived polypeptide thereof, contained in a plant extract having an essentially pure form or having said enzymatic activity. According to a transformed plant, part of a plant, plant cell or seed.

The present invention furthermore relates to a plant or part of a plant having enzymatic activity according to the present invention or a plant extract having the enzymatic activity as defined above or a recombinant DGL or a derivative thereof as defined above for use in the food field of humans or animals. It is about.

More specifically, the present invention relates to the use of a plant or part of a plant having an enzymatic activity according to the invention, in particular leaves, fruits, seeds as food.

In this aspect, the present invention specifically relates to a plant having the enzymatic activity or a part of a plant produced by the plant, in particular a food having a property for human or animal intake, including leaves or fruits or seeds. will be.

The invention also relates to one (or more) plant (s) and / or a part of the plant (s), in particular leaves and / or seeds and / or fruits and / or one (or), having said enzymatic activity. Or above) a plant extract (s) having said enzymatic activity and / or one (or more) recombinant polypeptide (s) of the invention, suitably in combination with one or more (or more) other ingestible compound (s). It relates to a nutritional composition.

The plant or part of the plant included in the nutritional composition is advantageously in the form of a powder.

The food according to the invention, the so-called functional food or the nutritional composition according to the invention are more particularly healthy individuals or individuals suffering from one or more lesions which may or may not affect the production level of the stomach and / or pancreatic lipase. It is intended to promote the absorption of animal or vegetable fats that are digested by. In this aspect the food or nutritional composition of the invention is advantageous for use as a nutritional supplement.

In addition, the present invention is directed to promoting absorption of animal or vegetable fats digested by healthy individuals or individuals suffering from one or more lesions that may or may not affect the level of production of gastrointestinal and / or pancreatic lipase. For the manufacture of a medicament (or pharmaceutical composition) a plant or part of a plant having an enzymatic activity according to the invention, in particular leaves and / or fruits and / or seeds, or plant cells, or having enzymatic activity as defined above Plant extracts, or the use of a recombinant polypeptide according to the invention, for example recombinant DGL or its derived polypeptide as defined above.

In particular, the pharmaceutical compositions are advantageous for use against individuals or older adults who are undergoing medical treatments that alter the fat absorption mechanism.

In addition, the pharmaceutical compositions according to the invention are more specifically for the treatment of lesions related to lipase (particularly gastrointestinal and / or pancreatic lipase) deficiencies in a subject, in particular lesions such as cystic fibrosis and pancreatic exocrine deficiency.

The present invention more specifically comprises pharmaceuticals comprising one (or more) plant extract (s) having said enzymatic activity and / or one or more (or more) recombinant polypeptides according to the invention, together with pharmaceutically acceptable excipients, where appropriate. It relates to a composition.

More specifically, the present invention relates to pharmaceutical compositions comprising recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) in essentially pure form or in the form of the enzyme extract.

Pharmaceutical compositions according to the invention are preferably administered orally, in particular in the form of capsules, tablets or powders for dilution.

The daily dosage for humans is preferably dispensed over the present mealtime as from about 200 mg to about 1,000 mg when the pharmaceutical composition comprises the enzyme extract, wherein the pharmaceutical composition is essentially pure according to the present invention. From about 100 mg to 500 mg in the form of a recombinant polypeptide.

The invention also relates to a plant or part of a plant having an enzymatic activity according to the invention, in particular leaves and (), for carrying out enzymatic reactions in industry in the field of fertilizers or in agro-related industries, in particular in the fat industry, local chemistry and dairy industries. Or) fruit and / or seeds, or plant cells, or plant extracts having the enzymatic activity as defined above, or the use of a recombinant polypeptide according to the invention, for example recombinant DGL or its derived polypeptide as defined above. .

In this aspect, the present invention relates to a plant or part of a plant or enzyme having an enzymatic activity according to the invention, in particular leaves and / or fruits and / or seeds, or plant cells, or plants having the enzymatic activity as defined above. One of the extracts, or industries in the field of fertilizers or agriculture-related industries, in particular in the local, local chemistry and dairy industries, carried out by extracts or recombinant polypeptides according to the invention, for example recombinant DGL or its derived polypeptides as defined above. The present invention relates to a method of bioconversion or biocatalysis by enzymes by performing the above enzyme reaction.

More specifically, the present invention can be used in the performance of the method and comprises one (or more) plant extract (s) having an enzymatic activity as defined above and / or one (or more) recombinant polypeptides according to the invention, In particular a recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4), if appropriate in combination with one (or more) additive (s) or other enzyme (s) that can be used for such industrial applications. The present invention relates to an enzyme preparation for fertilizer or industrial use in agriculture related industries.

The present invention more specifically relates to a plant having an enzymatic activity according to the present invention for carrying out a biomaterial conversion reaction or an enzymatic catalytic reaction by an enzyme, for example, an enzymatic hydrolysis or ester group transfer reaction on an industrial scale. Or part of a plant, in particular leaves, and / or fruits, and / or seeds, or the use of plant cells.

Plants or parts of plants, in particular leaves, and / or fruits, and / or seeds, or plant cells having enzymatic activity according to the invention are advantageously used as both an enzyme source and a reaction substrate.

The invention also relates to plants or parts of plants with enzymatic activity according to the invention, in particular leaves, and / or fruits, and / or seeds, or plant cells, in particular recombinants, which can be used both as an enzyme source and as a reaction substrate. A biocatalytic reaction using a plant comprising DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4).

The invention relates more particularly to the use of a plant or part of a plant having an enzymatic activity according to the invention for the manufacture of a biofuel.

In this aspect, the invention relates to alcohols, in particular methanol or ethanol, for powders of all or part of the transformed plants according to the invention, advantageously for the powders of transformed rapeseeds, sunflowers, soybean seeds according to the invention. The present invention relates to a method for producing a biofuel, which comprises the addition of and the recovery of the biofuel by filtration in particular.

The present invention furthermore relates to esters of plant fatty acids, in particular methyl esters of oleic acid, obtained by carrying out the process.

The present invention further relates to a biofuel obtained by carrying out the process, more particularly to the biofuel comprising esters of plant fatty acids.

More specifically, the present invention relates to a biofuel obtained by the performance of the method on rapeseed, more particularly said biofuel comprising a methyl ester of oleic acid.

The invention also relates to the use of said antibody directed against the recombinant polypeptide of the invention for carrying out a method of detecting or analyzing DGL or HGL in a biological sample.

More specifically, the present invention relates to the use of said antibody for carrying out an in vitro diagnostic method of a lesion related to an excess or vice versa insufficient or no production of lipase in a subject.

The in vitro diagnostic method performed on a biological sample taken from the patient consists of locating the sample with at least one antibody according to the invention followed by detection of the antibody-HGL complex formed during the detection step.

In view of the above, the present invention also

Reagents for the construction of said antibody advantageously labeled in a radiation or enzymatic manner and the medium advantageous for carrying out an immunological reaction between said antibody and HGL,

Reagents that allow detection of immunological complexes formed between the antibody and HGL

It relates to a kit for performing the in vitro detection or diagnostic method comprising a.

More specifically, the present invention provides on the one hand addition and / or inhibition of cDNA, or one (or more) nucleotide (s), shown in FIG. 2 encoding human gastrointestinal lipase (HGL), shown in FIG. Or) addition of a nucleotide sequence, or one (or more) amino acid (s), capable of encoding a polypeptide having an amino acid sequence identical to the amino acid sequence of HGL shown in FIG. 5, derived from the cDNA by substitution; and / or ) A nucleotide encoding a polypeptide having lipase activity derived from HGL by inhibition and / or substitution, on the other hand a component which causes the plant cell to produce a polypeptide encoded by said cDNA or said derived sequence, in particular A recombinant nucleus comprising a transcriptional promoter and a transcription terminator recognized by the transcriptional machinery of the plant cell (more specifically, the RNA polymerase of the plant cell) For transforming plant cells to obtain recombinant HGL in active enzyme form or one (or more) derived polypeptide (s) as defined above, from a transformed plant cell or a plant obtained from the cell, of the leotard sequence It is about a use.

In this aspect, the present invention provides the above-mentioned recombination in plant transformation for the production of recombinant DGL, wherein the nucleotide sequence shown in FIG. 1 or 3 encoding DGL is substituted with the nucleotide shown in FIG. 4 encoding HGL. It relates to a nucleotide sequence.

More specifically, the present invention relates to the following recombinant nucleotide sequences:

A promoter pSP of Agrobacterium tumefaciens, a sequence encoding a signal peptide of HGL, a nucleotide sequence shown in FIG. 4 immediately after this sequence, and a terminator polyA 35S of CaMV in the 5 '→ 3' direction Nucleotide sequence (called pSP-HGLSP-HGL),

A promoter pSP of Agrobacterium tumefaciens, a sequence encoding a signal peptide of HPL, a nucleotide sequence shown in FIG. 4 immediately after this sequence, and a terminator polyA 35S of CaMV in the 5 '→ 3' direction Nucleotide sequence (called pSP-HPLSP-HGL),

-The sequence encoding the promoter pSP of Agrobacterium tumefaciens, part of the signal peptide of RGL in the 5 '→ 3' direction, the nucleotide sequence shown in FIG. 4 immediately after this sequence, and the terminator polyA 35S of CaMV Nucleotide sequence, referred to as pSP-RGLSP-HGL.

The invention also relates to a vector comprising said recombinant polypeptide sequence encoding said HGL and / or derived polypeptide thereof and to a cell host transformed with said vector.

In addition, the present invention

Transforming the plant cell such that one (or more) recombinant nucleotide sequence (s) according to the invention is integrated into the genome of the plant cell,

If appropriate, generating a transformed plant from said transformed cells,

Lipase activity derived from said recombinant HGL by addition and / or inhibition and / or substitution of recombinant HGL and / or one (or more) amino acid (s) produced in said cell or said transformed plant. Recovering the polypeptide (s) having it, in particular by extraction and, if appropriate, by extraction after purification

Reactive HGL in the form of an active enzyme, and / or a method for producing a polypeptide (s) having lipase activity derived from recombinant HGL by the above method.

The present invention also relates to a method for producing recombinant HGL by performing the method described in the preparation of said recombinant DGL and / or its derived polypeptide using said recombinant sequence comprising the sequence shown in FIG.

As polypeptides derived from recombinant HGL obtainable in the practice of the method according to the invention, the following polypeptides may be mentioned:

A polypeptide consisting of the amino acids at positions 74 to 398 in FIG. 5 (Δ54HGL),

The polypeptide consisting of the amino acids at positions 24 to 398 in FIG. 5 (Δ4HGL).

More specifically, the present invention,

-Of Agrobacterium tumefaciens transformed by said plasmid comprising said recombinant nucleotide sequences pSP-HGLSP-HGL and / or pSP-HPLSP-HGL and / or pSP-RGLSP-HGL on a suitable culture medium. Transforming explant cells of the leaves of the plant by positioning them with the strain,

Selecting a transformed explant on a medium comprising kanamycin,

Production of the transformed plant from the explant by culturing the transformed explant on a suitable medium,

Recombinant HGL by recovery of the supernatant constituting the plant extract with milling, centrifugation and enzymatic activity of the leaves and / or seeds and / or fruits of said transformed plant in a suitable buffer, and, if appropriate, polypeptide (Δ54HGL) ) And / or extraction of the polypeptide (Δ4HGL),

-Where appropriate, from the extract obtained during this step, in particular, the supernatant is chromatographed to purify recombinant HGL and / or polypeptide (Δ54HGL) and / or polypeptide (Δ4HGL) to obtain recombinant HGL and / or polypeptide ( Preparing Δ54HGL) and / or polypeptide (Δ4HGL) in an essentially pure form

The method for producing the recombinant HGL and / or polypeptide (Δ54HGL) and / or polypeptide (Δ4HGL), characterized in that consisting of.

The invention also relates to a plant or part of a plant, in particular leaves, and / or fruit, comprising said one (or more) recombinant nucleotide sequence (s) integrated in its genome in a stable manner in accordance with the invention, and ( Or) seed.

The invention relates to one (or more) recombinant polypeptide (s) according to the invention, such as recombinant HGL and / or polypeptide (Δ54HGL) and / or polypeptide (Δ4HGL), having enzymatic activity, in particular lipase activity as defined below. It relates to a plant extract produced by the practice of the method of the present invention, including).

More specifically, the present invention is obtained by transformation of explant cells of a plant using the sequences pSP-HGLSP-HGL and / or pSP-HPLSP-HGL and / or pSP-RGLSP-DGL according to one of the above methods. Extracts of leaves and / or seeds of plants comprising recombinant HGL and / or polypeptide (Δ54HGL) and / or polypeptide (Δ4HGL), in particular

Extracts of tobacco leaves or seeds obtained by transformation of explant cells of tobacco leaves using the sequences pSP-HGLSP-HGL and / or pSP-HPLSP-HGL and / or pSP-RGLSP-HGL according to the above method And polypeptide (Δ54HGL) together with polypeptide (Δ4HGL), wherein the weight percent of the mixture of polypeptide (Δ54) and polypeptide (Δ4) is from about 0.1% to about 20% based on the total weight of protein present in the extract Extracts of tobacco leaves or seeds, wherein the extract has an enzyme activity of about 100 U / g (FW) to about 300 U / g (FW),

Extract of tobacco leaf obtained by transformation of explant cells of tobacco leaf using the sequence pSP-RGLSP-HGL according to the above method, wherein the polypeptide (Δ4HGL) is about 0.1 based on the total weight of the protein present in the extract An extract of tobacco leaves, comprising from% to about 20% by weight and the enzyme activity of the extract is from about 100 U / g (FW) to about 300 U / g (FW).

In addition, the invention is shown in FIG. 2, which is obtained in essentially pure form by carrying out one of the methods of the invention comprising the step of purifying the recombinant polypeptide of the invention by chromatography performed on the enzyme extract. The present invention relates to a polypeptide having enzymatic activity of the amino acid sequence recombinant DGL, or lipase activity derived therefrom.

In addition, the invention is shown in FIG. 5, which is obtained in essentially pure form by carrying out one of the methods of the invention comprising the step of purifying the recombinant polypeptide of the invention by chromatography performed on the enzyme extract. Polypeptides having lipase activity, particularly polypeptides (Δ54HGL), derived from the HGL by enzymatic activity recombinant HGL, or addition and / or inhibition and / or substitution of one (or more) amino acid (s) of an amino acid sequence and (Δ4HGL).

As noted above in recombinant DGL, the present invention also provides

-Polyclonal or monoclonal antibodies directed against the recombinant HGL or the derived polypeptides according to the invention and the above uses thereof,

On the basis of a plant, or part of a plant, in particular leaves and / or fruits and / or seeds, or plant cells or plant extracts or recombinant HGL or derived polypeptides thereof, as defined above according to the invention Food or nutritional compositions or pharmaceutical compositions for industrial purposes,

A recombinant HGL or a derived polypeptide thereof, or a plant, or part of a plant, in particular leaves and / or fruits and / or seeds, and / or plant cells or plant extracts according to the invention A biomaterial conversion reaction or biocatalysis by an enzyme carried out from, or a biofuel formulation.

The recombinant nucleotide sequence according to the present invention and a method for producing a transformed plant and a biofuel producing a recombinant polypeptide will be illustrated in the detailed description below.

1. Construction of chimeric gene encoding recombinant protein of gastrointestinal lipase of dog and expressed in leaves and seeds

I-A) Construction of chimeric genes encoding recombinant DGL and expressed in tobacco

Expression in tobacco leaves and seeds of genes encoding dog gastrointestinal lipase (DGL) requires the following regulatory sequences.

1. Dual structure promoter 35S (pd35S) of CaMV (Cauliflower mosaic virus).

This corresponds to the duplex of the sequence activating transcription and lies upstream of the TATA component of the native promoter 35S (Kay et al. 1987).

2. Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA that produces transcript 35S of the sequence of Cauliflower mosaic virus.

Construction of various plasmids using recombinant DNA technology is derived from pBIOC4 (Sambrook et al., 1989). Binary plasmids are derived from pGA492 (An et al., 1986) and contain, on their transport DNA, the following sequence originating from plasmid pTiT37 of Agrobacterium tumefaciene between the right and left interfaces:

Structural promoters of the nos gene encoding nopalin synthase (Depicker et al., 1982); Neomycin phosphotransferase II (bug and bug) that is deleted from the region of the first 8 codons whose start codon is methionine ATG and is fused to the sequence of the first 14 codons of the sequence encoding the nos gene (Depicker et al. (1982)) (Berg Berg), 1983), encoding the nptII gene encoding; A sequence encoding a nos gene without the first 14 codons; the region of the nos terminator (Depicker et al., 1982); Of the multiple cloning site (or polylinker) (HindIII-XbaI-SacI-HpaI-KpnI-ClaI-BglII) region preceding the gene of cat encoding chloramphenicol acetyltransferase and of the plasmid pTiA6 of Agrobacterium tumefaciene The terminal sequence of gene 6 (Liu et al., 1993). To substantially eliminate all sequences encoding the gene of cat, plasmid pGA492 was digested twice by SacI (restriction site of the polylinker) and ScaI (restriction site present in the sequence of the cat gene), followed by manufacturer's instructions. According to the enzyme T4 DNA polymerase (New England Biolabs). Ligation of 20 ng of the modified plasmid was carried out in a reaction medium containing 1 μl of buffered T4 DNA ligase × 10 (Amersham), 2.5 U of enzyme T4 DNA ligase (Amersham) for 16 hours at 14 ° C. It was performed at 10 μl. The bacterium Escherichia coli DH5α which was previously competent treated was transformed (Hanahan (1983)). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. Phosphorylated HindIII-EcoRI adapter (stratzen cloning system) was used to modify HindIII restriction sites of the plasmid DNA of the retained clones to EcoRI restriction sites. To perform this modification, 500 ng of the retained clone's plasmid DNA was digested by HindIII, dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim) and 1500 ng of HindIII-EcoRI DNA adapter according to the manufacturer's instructions. Co-precipitated at −80 ° C. for 30 minutes in the presence of 1/10 volume of 3M sodium acetate (pH 4.8) and 2.5 times volume of anhydrous ethanol. After centrifugation at 12000 g for 30 minutes, the precipitated DNA was washed with 70% ethanol, dried, immersed in 8 μl of water, stored at 65 ° C. for 10 minutes, and then 1 μl of buffered T4 DNA ligase. Ligation was carried out at 14 ° C. for 16 hours in the presence of × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham). After inactivating T4 DNA ligase at 65 ° C. for 10 minutes, the ligation reaction mixture was digested by EcoRI, purified by electrophoresis on 0.8% agarose gel, eluted (Sambrook et al., 1989), Precipitate at −80 ° C. for 30 minutes in the presence of 1/10 volume of 3M sodium acetate (pH 4.8) and 2.5 volume of absolute ethanol, centrifuge at 12000 g for 30 minutes, wash with 70% ethanol and dry Then, ligation was carried out as above. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (Bully and Dolly, 1979) and analyzed by enzymatic digestion by HindIII and EcoRI. The binary plasmid thus generated, having only the last 9 codons of the sequence encoding the cat gene and having a unique EcoRI site, was named pBIOC4.

Expression cassettes consisting of promoter pd35S and terminator polyA 35S were isolated using plasmid pJIT163Δ. Plasmid pJIT163Δ is derived from plasmid pJIT163 which is itself derived from plasmid pJIT60 (Guerineau and Mullineaux, 1993). Plasmid pJIT163 possesses an ATG codon between the HindIII and SalI sites of the polylinker. To inhibit ATG and obtain plasmid pJIT163Δ, plasmid pJIT163 DNA was digested twice with HindIII and SalI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sampbrook, 1989), 1/10 volume Precipitated at -80 ° C for 30 minutes in the presence of 3M sodium acetate (pH 4.8) and 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and Act according to the instructions with a Cleno enzyme (New England Biolabs), followed by 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) followed by 1 volume of chloroform: isoamyl alcohol (24: 1). Extract to remove protein, precipitate in the presence of 1/10 volume 3M sodium acetate (pH 4.8) and 2.5 volume anhydrous ethanol for 30 minutes at -80 ° C, centrifuge for 30 minutes at 12000 g, 70% Wash with ethanol, The mixture was finally ligated at 14 ° C. for 16 hours in the presence of 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham). The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (Visible and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. To isolate the expression cassette (SacI-XhoI fragment) consisting of promoter pd35S and terminator polyA 35S, the plasmid DNA of the retained clone PJIT163Δ was digested with SacI and XhoI. SacI-XhoI fragments with expression cassettes were purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook, 1989), and 1/10 volume of 3M sodium acetate (pH 4.8) and 2.5 volumes of anhydrous ethanol Precipitate at -80 ° C for 30 minutes in the presence, centrifuge at 12000g for 30 minutes, wash with 70% ethanol and dry, followed by Mung Bean nuclease enzyme (New England) according to manufacturer's instructions BioLabs). 200 ng of this purified insert was cloned into 20 ng of plasmid DNA of pBIOC4 digested by EcoRI and processed by enzyme brubin nuclease and dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim) according to the manufacturer's instructions It was. This ligation reaction was carried out at 14 ° C. in 20 μl in the presence of 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 5 U of enzyme T4 DNA ligase (Amersham). It was carried out for 16 hours. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μl / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC21.

Gastrointestinal lipase (DGL) in dogs is naturally synthesized in precursor form. Mature DGL protein consists of 379 amino acids. The complementary DNA of DGL was cloned into the BglII and SalI sites of the expression vector pRU303 to produce the vector pDGL5.303 described in WO94 / 13816. This was used to construct a binary plasmid pBIOC25 containing PS-DGL and pBIOC26 containing PPS-DGL, wherein a signal peptide (PS) or prepropeptide (PPS, ie N-terminal vacuole directional sequence of plant origin, respectively) was signaled. The sequence encoding the peptide) precedes the sequence encoding the mature DGL. The PS and PPS sequences, consisting of 23 and 37 amino acids, respectively, are the sequences of sporamin A, a storage protein of sweet potato tuber (Murakami et al., 1986; Matsukaa and Nakamura, 1991).

To facilitate fusion between the sequence of mature DGL and the sequence of directional signal PS or PPS, 2-oligodeoxynucleotide, 5 'cagg agatc TTG TTT GGA AAG CTT CAT CCC 3' ( HydIII containing BglII site in plasmid and complementary) Site) and the supplemental HindIII restriction site into the 4th and 5th codons of the mature DGL sequence by mutation by PCR using 5 'CAT ATT CCT CAG CTG GGT ATC 3' (containing the unique PvuII site in the plasmid). It was modified by introduction. Amplification of the BglIII-PvuII fragment by PCR was performed using 10 μl of buffered taq DNA polymerase × 10 (500 mM KCl, 100 mM Tris HCl, pH 9.0 and 1% Triton × 100), 6 μl of 25 mM MgCl ₂ , 3 μl. 10 mM dNTP (dATP, dCTP, dGTP and dTTP), 100 pM of each 2-oligodeoxynucleotide, 5 ng of matrix DNA (expression vector pRU303 comprising complementary DNA of DGL), 2.5 U Taq DNA polymer The reaction medium was carried out in 100 μl of lasse (promega) and 2 drops of petroleum jelly. DNA was denatured at 94 ° C. for 5 minutes, 1 minute denaturation at 94 ° C., 1 minute hybridization at 50 ° C., respectively, followed by extension reaction at 72 ° C. for 5 minutes. This PCR reaction was carried out in a DNA thermal cycler of PERKIN ELMER CETUS. Extract with chloroform and remove oil. The DNA fragment contained in the reaction medium was precipitated at -80 ° C for 30 minutes in the presence of 1/10 volume of 3M sodium acetate (pH 4.8) and 2.5 volumes of absolute ethanol, centrifuged at 12000g for 30 minutes, 70% Washed with ethanol, dried and digested with two restriction enzymes BglII and PvuII. Digested DNA fragments derived from PCR were purified by electrophoresis on 2% agarose gel, eluted (Sambrook, 1989), and 1/10 volume of 3M sodium acetate (pH 4.8) and 2.5 volumes of anhydrous ethanol Precipitated at -80 ° C for 30 minutes in the presence, centrifuged at 12000g for 30 minutes, washed with 70% ethanol, dried, digested twice with BglII and PvuII and purified by electrophoresis on 0.8% agarose gel It was ligated with plasmid DNA of vector pSGL5.303 which was eluted, precipitated in alcohol, dried and dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim) according to the manufacturer's instructions. The ligation was performed using 100 ng of dephosphorylated vector and 50 ng of digested DNA fragments derived from PCR amplification, using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham). ) Was carried out at 14 ° C. for 16 h in the reaction medium in which is present. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some plasmid DNA of retained clones were identified by sequencing by dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Parmacia. Introduction of the HindIII restriction site does not modify the genetic code of DGL. Indeed, the native DGL sequence AAA TTA (Lys-Leu) becomes AAG CTT (Lys-Leu). The resulting plasmid is named pBIOC22 and comprises the sequence of the corresponding mature DGL protein below.

LEU: first codon of mature DGL, AMB: stop codon

a. Construction of binary plasmid pBIOC25 containing PS-DGL

To suppress the sequence encoding the polypeptide Leu-Phe-Gly-Lys (first four amino acids) of the mature DGL protein, the plasmid pBIOC22 was partially digested by BglII, in general by HindIII. This sequence was then combined with 23 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG) which were fused to the first four codons of the sequence encoding the mature DGL protein (PS-first four codons of mature SGL). And a sequence encoding the signal peptide PS of CCC AAT CCA GCC CAT TCC). PS-first four codons of sequence mature DGL using plasmid pMAT103 according to the PCR amplification protocol described above (Matsuoka and Nakamura, 1991), two oligodeoxynucleotides 5 'cagg agatctg ATG AAA GCC TTC ACA CTC GC 3 And amplified by PCR with the aid of 5 'G ATG AAG CTT TCC AAA CAA GGA ATG GGC TGG ATT GGG CAG G 3'. After double enzymatic digestion by BGlII and HindIII, DNA fragments derived from PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook, 1989), and 1/10 volume of 3M sodium acetate precipitated at −80 ° C. for 30 minutes in the presence of (pH 4.8) and 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and pre-digested with BglII and HindIII Plasmid DNA of pBIOC22 purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook, 1989), precipitated with alcohol and dried and dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim) according to the manufacturer's instructions It was ligated to. Ligation was performed using 100 ng of the dephosphorylated vector and 50 ng of digested DNA fragments derived from PCR amplification using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Ae). Mercham) was performed at 10 ° C. for 16 hours in 10 μl of the reaction medium. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some plasmid DNA of retained clones were identified by sequencing by dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. Sequences of PS and mature DGL were cloned and their open reading frames were maintained. The cleavage sequence between the PS and mature DGL sequences is Ser-Leu. The resulting plasmid was named pBIOC23 . Starting from pBIOC23, BglII-XbaI fragments with PS-DGL sequences are isolated by double enzymatic digestion by BglII and XbaI, purified by electrophoresis on a 0.8% agarose gel, and eluted (Sambrook, 1989) Precipitated with alcohol and dried. This DNA fragment was ligated to plasmid DNA of pBIOC21 which was treated with the Cleno enzyme, the HindIII site was digested and treated with the Cleno and dephosphorylated by an alkaline phosphatase enzyme in the calf gut according to the manufacturer's instructions. Ligation was performed using 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 5U using 20 ng of the dephosphorylated vector and 200 ng of DNA fragment containing PS-DGL. 20 h of reaction medium in which the enzyme T4 DNA ligase (Amersham) is present was carried out at 14 ° C. for 16 h. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (Boy and Dolly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting clone was named pBIOC25 . The nucleotide sequence of the fragment encoding the recombinant protein PS-DGL was confirmed by sequencing by dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. The plasmid DNA of the binary vector pBIOC25 was introduced by direct transformation into strain LBA4404 of Agrobacterium turfasiene according to the method of Holster et al. (1978). The effectiveness of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

b. Construction of the binary plasmid pBIOC26 containing PPS-DGL

To inhibit the sequence encoding the polypeptide Leu-Phe-Gly-Lys of the mature DGL protein, the plasmid pBIOC22 was digested in whole by BglII and in part by HindIII. This sequence consists of 37 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT) fused to the sequence of the first four codons of the mature DGL protein (PPS-first four codons of mature DGL). CCA GCC CAT TCC AGG TTC AAT CCC ATC CGC CTC CCC ACC ACA CAC GAA CCC GCC) was substituted by the sequence encoding the signal peptide PPS. The PPS-first four codons of this sequence matured DGL were subjected to two oligodeoxynucleotides 5 'cagg agatctg ATG AAA GCC TTC ACA CTC GC 3' and 5 'G ATG AAG CTT TCC AAA CAA GGC GGG TTC GTG TGT GGT TG 3' Amplified by PCR using plasmid pMAT103 (Matsuoka and Nakamura, 1991) following the PCR amplification protocol with the help of. After double enzymatic digestion by BGlII and HindIII, DNA fragments derived from PCR amplification were purified by electrophoresis on a 2% agarose gel, eluted (Sambrook et al., 1989), and 1/10 volume of 3M sodium acetate ( pH 4.8) and precipitate for 30 minutes at -80 ° C in the presence of 2.5 volumes of absolute ethanol, centrifuge for 30 minutes at 12000g, wash with 70% ethanol, dry, then double digested with BglII and HindIII and 0.8 Purified by electrophoresis on% agarose gel, electroeluted, precipitated with alcohol and dried and ligated to plasmid DNA of pBIOC22 dephosphorylated by alkaline phosphatase enzyme (Boehringer Mannheim) in pediatric organs according to the manufacturer's instructions. The ligation was performed using 100 ng of dephosphorylated vector and 50 ng of digested DNA fragments derived from PCR amplification using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Ae 10% of the reaction medium in which Mercham) is present was carried out at 14 ° C. for 16 hours. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (Visible and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes.

Some plasmid DNA of retained clones were identified by sequencing by dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. Sequences of PPS and mature DGL were cloned and their open reading preems were maintained. The cleavage sequence between the two sequences is Ala-Leu. The resulting plasmid was named pBIOC24 . BglII-XbaI fragments with PPS-DGL sequences starting from pBIOC24 were isolated by double enzymatic digestion by BglII and XbaI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook, 1989) Precipitated with alcohol and dried. This DNA fragment was ligated to plasmid DNA of pBIOC21 treated with Clenow enzyme according to the manufacturer's instructions, digested at the HindIII site, treated with Clenow and dephosphorylated with an alkaline phosphatase enzyme (Behringer Mannheim) in the calf intestine. 2 μl of buffered T4 DNA ligase × 10 (Amersham) and 2 μl of 50% polyethyleneglycol 8000 and 5 U of enzyme T4 DNA using 20 ng of dephosphorylated vector and 200 ng of DNA fragment containing PPS-DGL 20 h of reaction medium in which ligase (Amersham) is present was carried out at 14 ° C. for 16 h. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was named pBIOC26 . The nucleotide sequence of the fragment encoding the recombinant protein PPS-DGL was confirmed by sequencing by dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The plasmid DNA of the binary vector pBIOC26 was introduced by direct transformation with strain LBA4404 of Agrobacterium tumefaciene (1978) according to Holster et al. The effectiveness of the clones obtained was confirmed by enzymatic digestion of the introduced plasmid DNA.

c. Construction of the RGLSP-DGL Containing Binary Plasmid pBIOC41

Rabbit gastrointestinal lipase is synthesized in the form of a precursor consisting of a signal peptide of 22 amino acids located at the NH ₂ -end and preceding the polypeptide sequence of the mature lipase. The full cDNA containing clone pJO101 encoding rabbit gastrointestinal lipase is nucleic acid and polypeptide derivative encoding the name rabbit gastrointestinal lipase of invention in European Patent Application No. 92 403 055.4, filed December 11, 1992 by Institut de Recherche Jouveinal, For use in the production of this polypeptide and in pharmaceutical compositions based thereon.

The arrangement of the polypeptide sequence of the dog gastrointestinal lipases and the polypeptide sequence of the precursor of the rabbit gastrointestinal lipase demonstrated that the sequence LFGK is present in two proteins. In the polypeptide sequence of rabbit lipase measured from purified natural protein, the first three residues L, F and G are absent and form part of the signal peptide of the 22 amino acids of RGL. As a result, the signal peptide of rabbit gastrointestinal lipase of this common three amino acids was fused to the mature protein sequence of dog gastrointestinal lipase. The polypeptide sequence consists of 19 amino acids MWVLFMVAALLSALGTTHG.

To suppress the sequence encoding the polypeptide Leu-Phe-Gly-Lys (first four amino acids) of the mature DGL protein, the plasmid pBIOC22 was digested in whole by BglII and in part by HindIII. This sequence is the signal peptide RGLSP (ATG TGG GTG CTT TTC ATG GTG GCA GCT TTG of rabbit gastrointestinal lipase of 19 amino acids fused to the amino acids of the first 4 codons (RGLSP-first 4 codons of mature DGL) of the mature DGL protein). CTA TCT GCA CTT GGA ACT ACA CAT GGT). Plasmid pJO101 with the help of two oligodeoxynucleotides 5 'agg agatct caacaATG TGG GTG CTT TTC ATG GTG 3' and 5 'G ATG AAG CTT TCC AAA CAA ACC ATG TGT AGT TCC AAG TG 3' And amplified by PCR. After double enzymatic digestion by BGlII and HindIII, DNA fragments originated by PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), and 1/10 volume of 3M sodium acetate precipitate at −80 ° C. for 30 minutes in the presence of (pH 4.8) and 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and then double digested with BglII and HindIII Purified by electrophoresis on 0.8% agarose gel, electroeluted, precipitated with alcohol and dried and ligated to plasmid DNA of pBIOC22 dephosphorylated by alkaline phosphatase enzyme (Boehringer Mannheim) in pediatric organs according to the manufacturer's instructions. Ligation was performed using 100 ng of the dephosphorylated vector and 50 ng of digested DNA fragments derived from PCR amplification using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Ae). Mercham) was performed at 10 ° C. for 16 hours in 10 μl of the reaction medium. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (Visible and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some plasmid DNA of retained clones were identified by sequencing by dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. The sequences of RGLSP and mature DGL were cloned keeping their open reading preems (ie they consisted of unique open reading frames). The cleavage sequence between the RGLSP and mature DGL sequences is Gly-Leu. The resulting plasmid was named pBIOC40 . BglII-XbaI fragments with RGLSPS-DGL sequences starting from pBIOC40 were isolated by double enzymatic digestion by BglII and XbaI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook, 1989) Precipitated with alcohol and dried. This DNA fragment was ligated to plasmid DNA of pBIOC21 treated with Clenow enzyme according to the manufacturer's instructions, digested at the HindIII site, treated with Clenow and dephosphorylated with alkaline phosphatase enzyme (Behringer Mannheim) in the calf intestine. Ligation was performed using 20 ng of the dephosphorylated vector and 200 ng of DNA fragment containing RGLSP-DGL. 20 μl of reaction medium in which 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 5 U of enzyme T4 DNA ligase (Amersham) are present for 16 hours at 14 ° C. Was performed. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was named pBIOC41 . The nucleotide sequence of the fragment encoding the recombinant protein RGLSP-DGL was confirmed by sequencing by dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The plasmid DNA of the binary vector pBIOC41 was introduced by direct transformation with strain LBA4404 of Agrobacterium tumefaciene (1978) according to Holster et al. The validity of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

I-B) Construction of chimeric genes encoding recombinant DGL and expressed in tomato

The constructs used are the same plasmids pBIOC25, pBIOC26 ALC pBIOC41, as used for the genetic transformation of tobacco.

II. Construction of Chimeric Gene Coding Recombinant Protein of Dog Gastrointestinal Lipase and Expressed in Rapeseed

a) Construction of the promoter pCRU containing binary plasmid pBIOC28

Expression of dog gastrointestinal lipase (DGL) in rapeseed required insertion of cDNA encoding DGL between the following regulatory sequences.

1. A promoter pCRU that corresponds to the non-coding region 5 'of the CRUCIFERIN A gene, the storage protein of radish seeds (Depigny-This et al., 1992), and which enables specific expression in seeds,

2. Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA that produces transcript 35S of the sequence of Cauliflower mosaic virus.

Promoter pCRU-containing fragments similar to pBIOC21 but with plasmid pBI221-CRURSP derived from pBI221 (commercially available by Defigni-Dys et al., 1992) to obtain a binary plasmid in which promoter pd35S is substituted by promoter pCRU EcoRI treated with Klenow-BamHI was isolated. By replacing promoter 35S with promoter pCRU, this plasmid pBI221-CRURSP is derived from pBI221 (commercially available from Clontech).

Fragment Klenow-BamHI treated EcoRI with promoter pCRU was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated with alcohol, dried, digested with KpnI and prepared Treated with T4 DNA polymerase (New England Biolabs), digested with BamHI, electrophoresed on 0.8% agarose gel, precipitated with alcohol, dried and calf intestinal alkaline phosphatase enzyme (Boringer) according to manufacturer's instructions Plasmid DNA of pJIT163 (described above) dephosphorylated by Mannheim). This ligation was performed in 20 μl of the reaction medium in which 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 5 U of enzyme T4 DNA ligase (Amersham) were present. It was carried out at 16 ℃ for 16 hours. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC27 .

Expression cassettes consisting of promoter pCRU and terminator polyA 35S were digested entirely by XhoI using pBIOC27 and partially digested by EcoRI. Purification by electrophoresis on 0.8% agarose gel, electroelute (Sambrook, 1989), precipitation with alcohol, drying, treatment with Klenow (New England Biolabs) according to manufacturer's instructions, manufacturer Was ligated to the EcoRI site of plasmid DNA of pBIOC24 dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim). This ligation was carried out at 14 ° C. in 20 μl of reaction medium in which 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 5 U of enzyme T4 DNA ligase (Amersham) were present. Was carried out for 16 h. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC28 .

b) Construction of the PPS-DGL containing binary plasmid pBIOC29

Isolation of BglII-XbaI fragments having the sequence PPS-DGL using pBIOC24 has already been described in IAb. This fragment was ligated to the EcoRI site of plasmid DNA of pBIOC28 treated with Clenow and dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim) according to the manufacturer's instructions. This ligation was performed using 20 ng of the dephosphorylated vector and 200 ng of BglII-XbaI DNA fragment containing PPS-DGL, 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethyleneglycol 8000 and 20 h of reaction medium in which 5 U of enzyme T4 DNA ligase (Amersham) is present was performed at 14 ° C. for 16 h. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC29 . The nucleotide sequence of the recombinant protein PPS-DGL was confirmed by sequencing by didideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The plasmid DNA of the binary vector pBIOC29 was introduced by direct transformation with strain LBA4404 of Agrobacterium tumefaciene (1978) according to Holster et al. The validity of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

c) Construction of binary plasmids pBIOC90 and pBIOC91 containing pGEA1D promoter

Expression of animal gene encoding dog gastrointestinal lipase (DGL) in rapeseed required the following regulator sequences.

1. Promoter pGEA1, which corresponds to the non-coding region 5 'of the gene of the storage protein GEA1 of Arabidopsis thaliana seeds (Gaubier et al., 1993) and enables specific expression in seeds,

2. Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA that produces transcript 35S of the sequence of Cauliflower mosaic virus.

Similar to pBIOC21, in order to obtain a binary plasmid pBIOC90 in which promoter pd35S was substituted by promoter pGEA1D, plasmid pGUS2-pGEA1 was used to isolate the fragment HindIII-BamHI containing promoter pGEA1 and treated with clenow. By substituting promoter 35S by promoter pGEA1, clone pGUS-2-pGEA1 derived from pBI221 contains two ATGs in frame, ATG of gene GEA1 (Em2) and ATG of gene gus. The ATG of gene GEA1 was destroyed. The DNA fragment contained between the SalI site and the upstream sequence of ATG of gene GEA1 of clone pGUS-2-pGEA1 was amplified using two oligonucleotides 5'CAAACGTGTACAATAGCCC 3 'and 5' CCCGGGGATCCTTTTTTG 3 '. Hybridization temperature was adjusted. Fragments amplified by PCR were digested by SalI and BamHI, purified by electrophoresis on 2% agarose gel, eluted (Sambrook et al., 1989), 1/10 volume of 3M sodium acetate (pH 4.8) And precipitated at −80 ° C. for 30 minutes in the presence of 2.5 volumes of absolute ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, double digested with SalI and BamHI and electrophoresis on 0.8% agarose gel. It was ligated to plasmid DNA of pGUS-2-GEA1 which was purified by electrophoresis, electroeluted, precipitated with alcohol and dried. The ligation was performed using 100 μg of vector and 50 ng of digested DNA fragments derived from the PCR amplification using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham). It was carried out for 16 hours at 14 ℃ in 10 μl of the reaction medium present. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Retained specific clones were identified by sequencing by didideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The resulting clone was named pGUS-2-pGEA1D .

HindIII-BamHI fragments with pGEA1D isolated from pGUS-2-pGEA1D and treated with Klenow according to the manufacturer's (BioLabs) instructions were purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook, 1989), ligated to alcohol, ligated to the KpnI and HindIII sites of plasmid DNA of pBIOC21 treated with T4 DNA polymerase (BioLabs) according to the manufacturer's instructions. This ligation was performed using 20 ng of dephosphorylated pBIOC21 and 200 ng of the XhoI-EcoRI DNA fragment, 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of enzyme 20 h of reaction medium in which T4 DNA ligase (Amersham) is present was carried out at 14 ° C. for 16 h. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (Boy and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC90 .

To obtain the binary plasmid pBIOC91, a HindIII fragment with the expression cassette p35S-nptII-tNOS described by Prom et al. (1986) was prepared at the HindIII site of pSCV1 produced by Edwards (Edwards GA, 1990) according to a conventional cloning method. The expression cassette pGEA1D-tNOS isolated from pBSII-pGEA1D was introduced into the binary plasmid pSCV1.2 obtained by cloning.

Plasmid pBSII-pGEA1D was prepared in two steps.

-On the one hand, a SacI-EcoRI fragment with tNOS (terminator of the nopaline synthase gene) of Agrobacterium tumefaciene treated and purified with the enzyme T4 DNA polymerase (BioLabs) was prepared according to the manufacturer's instructions. The instructions were cloned into the EcoRV site of pBSIISK + (stratogen) dephosphorylated by calf intestinal alkaline phosphatase enzyme (Boehringer Mannheim). Ligation was performed using 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of enzyme T4 using 20 ng of dephosphorylated vector and 200 ng of DNA fragment containing tNOS. 20 μl of reaction medium in which DNA ligase (Amersham) is present was carried out at 14 ° C. for 16 hours. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (Visible and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Retained specific clones were identified by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The resulting plasmid was named pBSII-tNOS .

On the other hand, fragments with pGEA1D which were double digested with HindIII, treated with the enzymes Klenow and BamHI and purified were cloned into XbaI and BamHI sites treated with Klenow of the plasmid pBSII-tNOS. The ligation was performed using 100 ng of the vector and 50 ng of the DNA fragment, 10 μl of the reaction medium in which 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham) were present. At 14 ° C. for 16 h. Bacterial Escherichia coli DH5α, previously competent, was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (Visible and Dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBSII-pGEA1D .

The expression cassette pGEA1D-tNOS carried by the Clenow treated XbaI-HindIII fragment was then cloned into the SmaI site of pSCV1.2 dephosphorylated by alkaline phosphatase enzyme in calf in accordance with the manufacturer's instructions. Ligation was performed using 20 ng of dephosphorylated pSCV1.2 and 200 ng of fragment with the expression cassette pGEA1D-tNOS using 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 20 h of the reaction medium in which the enzyme T4 DNA ligase (Amersham) is present was carried out at 14 ° C. for 16 hours. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC91 .

d) Construction of the binary plasmid pBIOC92 containing the pGEA6D promoter

Expression of animal gene encoding dog gastrointestinal lipase (DGL) in rapeseed required the following regulatory sequences.

1. Promoter pGEA6, corresponding to the non-coding region 5 'of the gene of the storage protein GEA6 of Arabidopsis thaliana seeds (Gaubier et al., 1993) and allowing specific expression in seeds,

2. Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA that produces transcript 35S of the sequence of Cauliflower mosaic virus.

To obtain a binary plasmid similar to pBIOC92 but with promoter pd35S substituted by promoter pGEA6D, plasmid pGUS2-pGEA6 was used to isolate the fragment EcoRI-BamHI containing pGEA6 and treated with klenow. The clone pGUS-2-pGEA6 derived from pUC18 contains two ATGs: ATG of gene GEA6 (Em6) and ATG of gene gus. The ATG of gene GEA6 was destroyed. DNA fragments contained between the AccI site and the upstream sequence of ATG of gene GEA6 of clone pGUS-2-pGEA6 were amplified using two oligonucleotides 5 'AAGTACGGCCACTACCACG 3' and 5 'CCCGGGGATCCTGGCTC 3'. Hybridization temperature was adjusted.

Fragments amplified by PCR were digested by AccI and BamHI, purified by electrophoresis on 2% agarose gel, eluted (Sambrook et al., 1989), 1/10 volume of 3M sodium acetate (pH 4.8) And precipitated at −80 ° C. for 30 minutes in the presence of 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and then double digested with AccI and BamHI and 0.8% agar. Purified by electrophoresis on Ross gel, ligated to plasmid DNA of pGUS-2-GEA6, eluted with alcohol and dried. Ligation was performed using 100 μg of the vector and 50 ng of digested DNA fragments derived from the PCR amplification, using 1 μl of buffered T4 DNA ligase × 10 (Amersham) and 2.5 U of enzyme T4 DNA ligase (Amersham). 10 μl of the reaction medium in which the reaction was carried out at 14 ° C. for 16 hours. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones, selected on 50 μg / ml of ampicillin, were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Retained specific clones were identified by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. The resulting clone was named pGUS-2-pGEA6D .

EcoRI-BamHI fragments with promoter pGEA6D isolated from pGUS-1-pGEA6D and treated with Klenow according to manufacturer's (BioLabs) instructions were purified by electrophoresis on 0.8% agarose gel, eluted (Sambrook) , 1989), precipitated with alcohol, dried, and then ligated to the XhoI site of plasmid DNA of the clenotreated modified pBIOC21. To delete the fragment with promoter pd35S and replace it with KpnI-EcoRV fragment with polylinker of KpnI-XhoI-SalI-ClaL-HindIII-BamHI-SmaI-EcoRI-EcoRV site of pBSIISK + instead Modified plasmid pBIOC21 was obtained by double digestion of pBIOC21 with HindIII treated with.

Ligation was performed using 20 ng of dephosphorylated pBIOC21 and 200 ng of EcoRI-BamHI DNA fragment, 2 μl of buffered T4 DNA ligase × 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of enzyme T4 DNA. 20 h of reaction medium in which ligase (Amersham) is present was carried out at 14 ° C. for 16 h. The previously competent bacterial Escherichia coli DH5α was transformed (Hanhan, 1983). Plasmid DNA of the resulting clones selected on 12 μg / ml tetracycline were extracted by alkaline lysis (visible and dolly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was named pBIOC92 .

e) Construction of RGLSP-DGL containing binary plasmids pBIOC93, pBIOC94, pBIOC95, pBIOC96 and pBIOC97

Plasmid pBIOC40 is as described above. This plasmid contains fragment BglII-XbaI having the sequence RGLSP-DGL.

This fragment was isolated by double enzymatic digestion by BglII and XbaI, purified by electrophoresis on 0.8% agarose gel, electroeluted, precipitated with alcohol, dried, treated with Klenow and then treated with Klenow PBIOC28 digested and dephosphorylated by treated EcoRI (described above), pBIOC90 digested and dephosphorylated by EcoRI treated with Klenow, pBIOC91 digested and dephosphorylated by SmaI, treated with Klenow HindIII PBIOC92 digested and dephosphorylated by pBIOC92, respectively, to produce pBIOC93, pBIOC94, pBIOC95 and pBIOC96, respectively.

Plasmid pBIOC97 was purified by treatment of fragment KpnI-EcoRV with expression cassette pGEA6D-RGLSP-DGL-t35S with enzyme T4 DNA polymerase, electrophoresis on 0.8% agarose gel, electroeluted, precipitated with alcohol, After drying, it is produced by ligation to ligation to pSCV1.2 digested with SmaI and dephosphorylated. The expression cassette pGEA6D-RGLSP-DGL-t35S is derived from pBIOC92.

III. Construction of chimeric genes encoding recombinant proteins of human gastrointestinal lipases, for example enabling constitutive expression in tobacco leaves and tobacco seeds.

a) Construction of the binary plasmid pBIOC82 comprising the chimeric promoter, SUPER-PROMOTER pSP.

To express genes encoding human gastric lipase (HGL) in tobacco leaves, the following regulatory sequences were required:

1. Super-promoter, a chimeric promoter (pSP; PCT / US94 / 12946). The promoter is a fusion of three transcriptional activator components of the agrobacterium tumefaciens octopin synthase gene promoter, a transcriptional activator component of the agrobacterium tumefaciens mannose synthase gene promoter and a mannofin synthase promoter It is configured by.

2. Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA that produces transcript 35S of the sequence of Cauliflower mosaic virus.

To obtain a binary plasmid similar to pBIOC21, replacing promoter pd35S with promoter pSP, pvuII-SalI fragments containing promoter pSP, reacted with Klenow, were isolated using plasmid pBISNI (PCT / US94 / 12946). Purified by electrophoresis on 1% agarose gel, electroeluted (Sambrook et al., 1989), precipitated with alcohol and dried, treated with DNA T4 polymerase, double digested with KpnI and EcoRI and calf as directed by the manufacturer. It was ligated to pBIOC81 plasmid DNA dephosphorylated with enteric alkaline phosphatase enzyme (Boehringer Mannheim). The pBIOC81 plasmid corresponds to the XbaI position of deleted pBIOC21. To accomplish this, the pBIOC21 plasmid was cleaved with XbaI and then treated with Klenow enzyme to ligate under the action of T4 DNA ligase.

2 μl of T4 DNA ligase (Amersham), 2 μl of 50% polyethylene glycol 8000 and T4 DNA ligase enzyme using 20 ng of the dephosphorylation vector described above and 200 ng of DNA fragment with pSP described above Mersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 h. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 12 μg / ml tetracycline were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting plasmid was named pBIOC82.

Human gastrointestinal lipase (HGL) is naturally synthesized in the form of precursors, as described in a 1987 publication by Bodmer et al. Mature HGL protein consists of 379 amino acids. The signal peptide (HGLSP) of the protein consists of 19 amino acids. The restriction enzyme position between HGLSP and HGL is Gly-Leu.

The natural signal peptide HGLSP, the signal peptide of human pancreatic lipase (HPLSP; Giller et al., 1992) and the signal peptide of rabbit gastrointestinal lipase (RGLSP; already described previously; European Patent Application No. 92.403055.4, respectively, before the sequence encoding HGL) Sequences encoding HGL precursors were used to construct binary plasmids of pBIOC85 comprising HGLSP-HGL, pBIOC87 comprising HPLSP-HGL, and pBIOC89 comprising RGLSP-HGL.

Sequences encoding precursors of HGL were isolated by double digestion with PstI and DraI, purified by electrophoresis on 0.8% agarose gel, eluted (Sambrook et al., 1989), 3 M sodium acetate with pH 4.8 Precipitated at −80 ° C. for 30 minutes in the presence of 1/10 volume and 2.5 volumes of absolute ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol and dried. The sequence was then cloned into the PstI and SpeI positions of the pBSIISK + plasmid sold by Stratagene (T4 DNA polymerase (BioLabs) enzymes were allowed to function as instructed by the manufacturer). Using 100 ng of vector and 50 ng of DNA fragment having the sequence encoding the precursor of HGL described above, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) 2.5 U Ligation was carried out at 10 ° C. for 16 h in 10 μl of reaction medium in the presence of. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 50 μg / ml ampicillin were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting plasmid was named pBIOC83.

5 'aaactgcaggctcgag TTG TTT GGA AAA TTA CAT CCT GGA tcc CCT GAA GTG ACT ATG 3' (including unique PstI, XhoI and BamHI positions) and 5 'AAT GGT GGT GCC CTG GGA ATG GCC AAC ATA GTG TAG CTG C 3' Direct mutagenesis was carried out by PCR using two oligodeoxynucleotides (including the unique MscI position in the pBIOC83 plasmid) to introduce previously unexistent BamHI positions into the ninth and tenth codons. Thereby altering the sequence encoding the mature HGL.

10 μl of 10-fold Taq DNA polymerase buffer (500 mM KCl, 100 mM Tris-HCl with pH 9.0 and 1% Triton x100), 6 μl 25 mM MgCl ₂ , 10 mM dNTP (dATP, dCTP, dGTP and dTTP) 3 μl, 100 pM each of the two oligodeoxy-nucleotides described above, 5 ng of matrix DNA (pBIOC83 vector), 2.5 U of Taq DNA polymerase (Promega) and 2 drops of petrolatum PstI- in a reaction medium of 100 μl. MscI fragments were amplified by PCR. DNA was denatured at 94 ° C. for 5 minutes, followed by 30 cycles of 1 minute of denaturation at 94 ° C., 1 minute of hybridization at 65 ° C. and 1 minute of elongation at 72 ° C., followed by 72 minutes at 72 ° C. The kidneys lasted for 5 minutes. This PCR reaction was carried out in a DNA thermal cycler from Perkin Elmer Setus. The oil was removed by extraction with chloroform. The DNA fragments contained in the reaction medium were then precipitated at −80 ° C. for 30 minutes in the presence of 1/10 volume of 3 M sodium acetate having a pH of 4.8, and 2.5 volumes of anhydrous ethanol, and centrifuged at 12000 g for 30 minutes. Washed with 70% ethanol, dried and cleaved with two restriction enzymes, PstI and MscI. Cleaved DNA fragments derived from PCR amplifications were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), 1/10 volume of 3 M sodium acetate at pH 4.8, and anhydrous ethanol 2.5 Precipitated at −80 ° C. for 30 minutes in the presence of volume, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and then cut twice with PstI and MscI and electrophoresed on 0.8% agarose gel. Purified by electrolysis, precipitated with alcohol and ligated to dried pBIOC83 plasmid DNA. In the presence of 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) using 100 ng of vector and 50 ng of cut DNA fragments derived from the PCR amplifications described above. Ligation was performed for 16 hours at 14 ° C. in 10 μl of reaction medium. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 50 μg / ml ampicillin were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. Using the T7 (registered trademark) sequencing kit sold by Pharmacia, the sequencing was performed by the dideoxynucleotide method (Sanger et al., 1977) to identify some of the clones retained. The introduction of the BamHI restriction site does not alter the genetic code of HGL. In fact, the native HGL sequence GGA AGC (Gly-Ser) becomes the GGA TCC (Gly-Ser). The resulting plasmid was named pBIOC84.

b) Construction of binary plasmid pBIOC85 comprising HGLSP-HGL.

PstI-XbaI fragments with sequences of HGLSP-HGL were isolated by enzymatically cleaving pBIOC83 twice with PstI and XbaI, purified by electrophoresis on 0.8% agarose gel, and electroeluted (Sambrook et al., 1989). Precipitated with alcohol and dried. This DNA fragment was then treated with T4 DNA polymerase enzyme (BioLabs) as directed by the manufacturer, treated with Klenow (BioLabs) and dephosphorylated with calf pancreatic alkaline phosphatase enzyme (Behringer Mannheim) as directed by the manufacturer. And pBIOC82 plasmid DNA digested at the EcoRI site. Using 20 ng of the dephosphorylation vector and 200 ng of the DNA fragment containing the HGLSP-HGL, 2 μl of T4 DNA ligase (Amersham) 2 μl, 50% polyethylene glycol 8000 2 μl and T4 DNA ligase enzyme ( Amersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 hours. The Escherichia coli DH5α strain, previously competent, was transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 12 μg / ml tetracycline were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting clone was named pBIOC85. The nucleic acid sequence of the fragment encoding the HGLSP-HGL recombinant protein was confirmed by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The restriction sequence between HGLSP and mature HGL is Gly-Leu. The binary vector pBIOC85 plasmid DNA was introduced by direct transformation into strain Agrobacterium tumefaciens LBA4404 according to Holsters et al. (1978). The effectiveness of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

c) Construction of the binary plasmid pBIOC86 comprising HPLSP-HGL.

The pBIOC84 plasmid was cleaved twice with PstI and BamHI to inhibit the sequence encoding the signal peptide HGLSP and the first eight amino acids of the mature HGL protein (Leu-Phe-Gly-Lys-Leu-His-Pro-Gly). The sequence is the sequence encoding the 16 amino acids of the HPLSP signal peptide fused to the sequence encoding the first 8 codons of the mature HGL protein (ATG CTG CCA CTT TGG ACT CTT TCA CTG CTG CTG GGA GCA GTA GCA GGA) ( First 8 codons of HPLSP-mature HGL). Following the PCR amplification protocol described previously (see paragraph I above), 5 'aaactgcaggctcgagaacaATG CTG using two oligodeoxynucleotides of 5' aaactgcaggctcgagaacaATG C 3 'and 5' C AGG gga TCC AGG ATG TAA TTT TCC 3 ' CCA CTT TGG ACT CTT TCA CTG CTG CTG GGA GCA GTA GCA GGA TTG TTT GGA AAA TTA CAT CCT GGA tcc CCT G 3 'Matrix Amplified the first eight codon sequences of HPLSP-mature HGL by PCR. Hybridization temperature was adjusted. After two enzymatic cleavage by PstI and BamHI, DNA fragments derived from PCR amplification were electrophoretically purified on a 2% agarose gel, electroeluted (Sambrook et al., 1989), 3 M sodium with a pH of 4.8 Precipitated at −80 ° C. for 30 minutes in the presence of 1/10 volume of acetate and 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and then twice with PstI and BamHI. The cleavage was purified by electrophoresis on a 0.8% agarose gel, ligated to pBIOC84 plasmid DNA which was eluted (Sambrook et al., 1989), precipitated with alcohol and dried. Using 100 ng of vector and 50 ng of cut DNA fragments derived from the PCR amplification described above, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) 2.5 U Ligation was carried out in 10 μl of reaction medium at 16 ° C. for 16 hours. The Escherichia coli DH5α strain, previously competent, was transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 50 μg / ml ampicillin were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. Several of the retained clones were identified by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. HPLSP and mature HGL sequences that maintain an open reading frame have been cloned (ie, make up a unique open reading frame). The restriction sequence between the HPLSP sequence and the mature HGL sequence is Gly-Leu. The resulting plasmid was named pBIOC86.

PstI-XbaI fragments with HPLSP-HGL sequences were isolated by enzymatically cleaving BIOC86 twice with PstI and XbaI, purified by electrophoresis on 0.8% agarose gel, and eluted (Sambrook et al., 1989), Precipitated with alcohol and dried. This DNA fragment was then treated with T4 DNA polymerase (BioLabs) as directed by the manufacturer, treated with Klenow (BioLabs) and dephosphorylated with calf pancreatic alkaline phosphatase enzyme (Boehringer Mannheim) as directed by the manufacturer. PBIOC82 plasmid DNA digested at the EcoRI site was ligated. Using 20 ng of the dephosphorylation vector and 200 ng of DNA fragment containing the HPLSP-HGL, 2 μl of T4 DNA ligase (Amersham) 2 μl, 50% polyethylene glycol 8000 2 μl and T4 DNA ligase enzyme ( Amersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 hours. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). The plasmid DNA of clones obtained by selection on a medium containing 12 μg / ml tetracycline were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting clone was named pBIOC87. The nucleic acid sequence of the fragment encoding the HPLSP-HGL recombinant protein was confirmed by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The binary vector pBIOC87 plasmid DNA was introduced by direct transformation into the Agrobacterium tumefaciens LBA4404 strain according to Holsters et al. (1978). The effectiveness of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

d) Construction of the binary plasmid pBIOC89 comprising RGLSP-HGL.

The pBIOC84 plasmid was cleaved twice with PstI and BamHI to inhibit the sequence encoding the signal peptide HGLSP and the first eight amino acids of the mature HGL protein (Leu-Phe-Gly-Lys-Leu-His-Pro-Gly). The sequence encodes the 19 amino acids of the RGLSP signal peptide fused to the sequence encoding the first 8 codons of the mature HGL protein (ATG TGG GTG CTT TTC ATG GTG GCA GCT TTG CTA TCT GCA CTT GGA ACT ACA CAT GGT ) (The first eight codons of RGLSP-mature HGL). Following the previously described PCR amplification protocol (see paragraph I above), 5 'aaactgcaggctcgagaacaATG TGG using two oligodeoxynucleotides of 5' aaactgcaggctcgagaacaATG TGG 3 'and 5' C AGG gga TCC AGG ATG TAA TTT TCC 3 ' GTG CTT TTC ATG GTG GCA GCT TTG CTA TCT GCA CTT GGA ACT ACA CAT GGT TTG TTT GGA AAA TTA CAT CCT GGA tcc CCT G 3 'Matrix Amplified the first eight codon sequences of RGLSP-mature HGL by PCR. Hybridization temperature was adjusted. After double enzymatic cleavage by PstI and BamHI, DNA fragments derived from PCR amplifications were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), 3 M sodium acetate 1 with a pH of 4.8 Precipitated at −80 ° C. for 30 minutes in the presence of 10 volumes and 2.5 volumes of anhydrous ethanol, centrifuged at 12000 g for 30 minutes, washed with 70% ethanol, dried and then cut twice with PstI and BamHI. Purified by electrophoresis on 0.8% agarose gel and eluted (Sambrook et al., 1989), ligated to pBIOC84 plasmid DNA, precipitated with alcohol and dried. Using 100 ng of vector and 50 ng of cut DNA fragments derived from the PCR amplification described above, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) 2.5 U Ligation was carried out in 10 μl of reaction medium at 16 ° C. for 16 hours. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 50 μg / ml ampicillin were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. Several of the retained clones were identified by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using a T7® sequencing kit sold by Pharmacia. RGLSP and mature HGL sequences that maintain an open reading frame were cloned (ie, the sequences constitute a unique open reading frame). The restriction sequence between the RGLSP sequence and the mature HGL sequence is Gly-Leu. The resulting plasmid was named pBIOC88.

PstI-XbaI fragments with sequences of RGLSP-HGL were isolated by enzymatically cleaving pBIOC88 twice with PstI and XbaI, purified by electrophoresis on 0.8% agarose gel, and eluted (Sambrook et al., 1989). Precipitated with alcohol and dried. This DNA fragment was then treated with T4 DNA polymerase enzyme (BioLabs) as directed by the manufacturer, treated with Klenow (BioLabs) and dephosphorylated with calf pancreatic alkaline phosphatase enzyme (Behringer Mannheim) as directed by the manufacturer. And pBIOC82 plasmid DNA digested at the XbaI position. Using 20 ng of the dephosphorylation vector and 200 ng of DNA fragment containing RGLSP-HGL, 2 μl of T4 DNA ligase (Amersham) 2 μl, 50 μl polyethylene glycol 8000 2 μl and T4 DNA ligase enzyme Mersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 h. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Plasmid DNA of clones obtained by selection on 12 μg / ml tetracycline were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting clone was named pBIOC89. The nucleic acid sequence of the fragment encoding the RGLSP-HGL recombinant protein was confirmed by sequencing by the dideoxynucleotide method (Sanger et al., 1977) using the T7® sequencing kit sold by Pharmacia. The binary vector pBIOC89 plasmid DNA was introduced directly into the Agrobacterium tumefaciens LBA4404 strain according to Holsters et al. (1978). The effectiveness of the retained clones was confirmed by enzymatic digestion of the introduced plasmid DNA.

IV. Construction of chimeric genes encoding recombinant proteins of dog gastrointestinal lipases and expressed in corn seeds.

a) Construction of pBIOC98 and pBIOC99 plasmids containing RGLSP-DGL and allowing constitutive expression in corn seeds.

In order to constitutively express the animal gene encoding dog gastrointestinal lipase (DGL) in corn seeds, the following regulatory sequences were required:

1. One of the following two promoters to enable constitutional expression:

A rice actin promoter (pAR-IAR) followed by a rice actin intron contained in the pAct1-F4 plasmid described by McElroy et al. (1991);

-Dual Constitutive Promoter 35S (pd35S) of CaMV (Cabbage Mosaic Virus). This promoter corresponds to a duplex of sequences that activates transcription located upstream from the TATA component of the native 35S promoter (Kay et al., 1987);

2. One of two terminators

Terminator polyA 35S (Fanck et al., 1980), which is a terminal transcriptional sequence corresponding to the 3 'non-coding region of double-stranded circular DNA producing the transcript 35S of the sequence of Cauliflower mosaic virus.

Terminator polyA NOS, a terminal transcriptional sequence corresponding to the 3 'noncoding sequence region of the nopalin synthase gene of the Ti plasmid of the Agrobacterium tumefaciens nopalin strain (Depicker et al., 1982).

Cloning the BglII-XbaI fragment with the sequence encoding RGLSP-DGL at the NcoI and SalI sites of pBSII-pAR-IAR-tNOS to obtain a pBIOC98 plasmid in which the sequence encoding RGLSP-DGL is located under the control of pAR-IAR. .

BglII-XbaI fragments comprising sequences encoding RGLSP-DGL were isolated from pBIOC40 by enzymatic cleavage twice with BglII and XbaI (as described above) and purified by electrophoresis on 0.8% agarose gel and electrophoresis. Eluted, precipitated with alcohol and dried, then treated with Klenow enzyme. The pBSII-pAR-IAR-tNOS plasmid was digested twice with SalI and NcoI, purified, treated with brubin nuclease enzyme (BioLabs) and with calf intestinal alkaline phosphatase enzyme (Beringer Mannheim) Dephosphorylation. 2 μl of T4 DNA ligase (Amersham) 2 μl, 50% polyethylene glycol 8000 2 μl and T4 DNA lig using 20 ng of dephosphorylation vector and 200 ng of DNA fragment comprising the sequence encoding the RGLSP-DGL Ligation was performed for 16 hours at 14 ° C. in 20 μl of the reaction medium in the presence of 5 U of the first enzyme (Amersham). Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). The plasmid DNA of clones obtained by selection in a medium containing 50 μg / ml of ampicillin were extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting plasmid was named pBIOC98.

The pBSII-pAR-IAR-tNOS plasmid was treated with KlenI and KpnI of pBSII-tNOS of the SnaBI-KpnI fragment having a sequence corresponding to the pAR-IAR-initiation of the sequence encoding the gus gene isolated from the pActl-F4 plasmid Obtained by cloning to an Eco01091 site. Using 100 ng of vector and 50 ng of the DNA fragment, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 2.5 μl of T4 DNA ligase enzyme (Amersham) at 2.5 ° C. in 10 μl of the reaction medium at 14 ° C. Ligation was carried out for 16 hours. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Clonal plasmid DNA obtained by selection on a medium containing 50 μg / ml of ampicillin was extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes.

The pBSII-tNOS plasmid was isolated from pBI121 sold by Clontech and purified by electrophoresis on 2% agarose gel by double enzymatic digestion by SacI and EcoRV at the dephosphorylated EcoRV site of pBSIISK + sold by stratagen. Obtained by cloning the SacI-EcoRI fragment with the tNOS sequence treated with the polymerase enzyme. Using 20 ng of dephosphorylated vector and 200 ng of DNA fragment containing the tNOS sequence, 2 μl of T4 DNA ligase (Amersham) 2 μl, 50 μl polyethylene glycol 8000 2 μl and T4 DNA ligase enzyme Mersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 h. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Clonal plasmid DNA obtained by selection on a medium containing 50 μg / ml of ampicillin was extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes.

Cloning the KpnI-BamHI fragment having the sequence corresponding to pd35S-RGLSP-DGL isolated from pBIOC41 to the KpnI and BamHI sites of the pBSII-t35S plasmid to obtain a pBIOC99 plasmid in which the sequence encoding RGLSP-DGL is under pd35S control . Using 100 ng of vector and 50 ng of the DNA fragment, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 10 μl of reaction medium in the presence of 2.5 U of T4 DNA ligase enzyme (Amersham) 16 at 14 ° C. Ligation was carried out for hours. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Clonal plasmid DNA obtained by selection on a medium containing 50 μg / ml of ampicillin was extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes.

A cleavage of the pBSIISK + plasmid sold by Stratagen Co., Ltd., a SmaI-EcoRV fragment (described above) with the t35S sequence isolated from pJIT163 in two cleavages by the SmaI and EcoRV enzymes and electrophoretically purified on a 2% agarose gel. PBSII-t35S plasmids were obtained by cloning to dephosphorylated SpeI sites treated with nou. Using 20 ng of dephosphorylation vector and 200 ng of DNA fragment comprising the t35S sequence, 2 μl of T4 DNA ligase (Amersham) 2 μl, 50 μl polyethylene glycol 8000 2 μl and T4 DNA ligase enzyme Mersham) ligation was carried out in 20 μl of reaction medium in the presence of 5 U at 14 ° C. for 16 h. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Clonal plasmid DNA obtained by selection on a medium containing 50 μg / ml of ampicillin was extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes.

b) Construction of the pBIOC100 and pBIOC101 plasmids containing RGLSP-DGL and RGLSP-DGL-KDEL, respectively, and allowing expression in the milk of corn seeds

The following regulatory sequences were required to express the animal gene encoding dog gastrointestinal lipase (DGL) in the milk of corn seeds:

1. γzeine gene promoter (pγzeine) in maize contained in the pγ63 plasmid described by Reina et al. (1990). Cloning of pγzeine at the HindIII and XbaI sites of the pUC18 plasmid containing the p35S-gus-tNOS expression cassette of pBI221 sold by Clontech Co. between HindIII and EcoRI sites yielded a pγ63 plasmid. The plasmid is expressed in the milk of corn seeds.

2. Terminator polyA NOS, a terminal transcriptional sequence corresponding to the 3 'noncoding sequence region of the nopalin synthase gene of the Ti plasmid of the Agrobacterium tumefaciens nopalin strain (Depicker et al., 1982).

The sequence encoding RGLSP-DGL under pγzeine control by cloning XbaI-BglII fragments (described above) treated with Klenow isolated from pBIOC40 to the SacI and BamHI sites treated with T4 DNA polymerase enzyme of the pγ63 plasmid Obtained pBIOC100 plasmid was obtained. Using 100 ng of vector and 50 ng of the DNA fragment, 1 μl of 10-fold buffer of T4 DNA ligase (Amersham) and 10 μl of reaction medium in the presence of 2.5 U of T4 DNA ligase enzyme (Amersham) 16 at 14 ° C. Ligation was carried out for hours. Escherichia coli DH5α bacteria, previously competent, were transformed (Hanahan, 1983). Clonal plasmid DNA obtained by selection on a medium containing 50 μg / ml of ampicillin was extracted by alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion by restriction enzymes. The resulting clone was named pBIOC100.

Substituting pBIOC101 by replacing an NcoI-AflII fragment of pBIOC100 with an NcoI-AflII fragment having a sequence (directed to the endoplasmic reticulum) encoding a KDEL tetrapeptide located in front of the stop codon obtained by PCR amplification according to the protocol described above Obtain the plasmid. The two oligodeoxynucleotides used during the reaction were 5 'AAT CAC TTG GAC TTT ATC TGG Gcc atg gAT GCC 3' (specific NcoI site) and 5 'ATT ctt aag AAA CTT TAT TGC CAA ATG TTT GAA CGA TCG GGG AAA TTC GAC GCG TCT AGA ACT ATA GCT CAT CCT TAT TAT CTG TTC CCA TCA TGG 3 '(sequence encoding KDEL and unique AflII sites). Hybridization temperature was 70 ° C. Cloning was performed as previously described.

V. Preparation of Transgenic Flats

Seeds of spring rapeseed (Brassica napus cv WESTAR or Limagrain stock) are sterilized for 40 minutes in a 15% Domestos solution. After 4 washes with sterile water, 7 cm in diameter and height in Murashige and Skoog mineral medium (Sigma M 5519), which had 30 g / l sucrose and solidified with 5 g / l agar gel Seeds are germinated in a quantity of 20 seeds per pot of 10 cm. The pot is placed in a growth chamber at 26 ° C. with a photoperiod of 16 hours / 8 hours under a luminous intensity of 80 μE m ⁻² S ⁻¹ .

After 5 days of germination, the cotyledons are removed by slicing all of the leaf spigots about 1 mm above the cotyledons under sterile conditions.

At the same time, agrobacte comprising a pGAZE plasmid with a sequence encoding a dog gastrointestinal lipase fused to a sequence encoding a pBIOC29 (or pBIOC25), ie a directed signal PPS (or PS) under the control of a pCRU (or pd35S) promoter. Leeum tumefaciens strain LBA4404 is preincubated for 36 hours at 28 ° C. in a 50 ml conical flask containing 10 ml of bacterial medium 2YT (Sambrook et al., 1989) supplemented with antibiotics that can be used for selection of the strains used. .

The preculture is used to inoculate in a 1% amount in a new bacterial culture prepared under the same conditions. After 14 hours, the culture is centrifuged at 3000 g for 15 minutes and suspended in the same volume of germination liquid medium. This suspension is divided into 5 ml / dish in 5 cm diameter Petri dishes.

The cut end of the leaf stalk is immersed for several seconds in the Agrobacteria solution prepared as described above, and then the leaf stalk is pushed several millimeters into the regeneration medium. The medium has the same basic composition as the germination medium to which the benzyl-amino-purine (BAP), a plant hormone that promotes neoformation of shoots, is added at 4 mg / l. Ten explants (cotyledons with leafy stems) are grown per Petri dish (Greiner Ref. No. 664102) 9 cm in diameter.

After 2 days of incubation under the same environmental conditions as for germination, 45 mg / l of the screening agent kanamycin sulfate (Sigma, Ref. K4000) and 1/6 (by weight) of potassium salt of clavulanic acid for inhibiting bacterial development Eating out into a Phytatray box (Sigma, Ref. P1552) containing the previous medium supplemented with a mixture of 5/6 sodium salt of amoxicillin (Augmentin, injectable) in an amount of 600 mg / l Transfer the plant.

The explants are transplanted under sterile conditions on fresh medium under the same conditions for subsequent two transplants at three week intervals.

Individually in a transparent pot 5 cm in diameter and 10 cm high containing the same medium as the previous medium, except that the last green shoots appearing from the second or third transfer were separated from the explants and lacked BAP. To grow. After three weeks of growth, the stems of the transformed shoots are cut and transferred to the pot of fresh medium. After 3-4 weeks, the roots are fully developed so that the seedlings are acclimated to the new environment in the plant growth control room. Remove shoots that are not green or have no root. The seedlings are then planted into a 7 cm dish filled with water saturated soil (standard NF U44511: 40% brown peat, 30% sieved heat and 30% sand). After two weeks of new environmental acclimation in a plant growth control room (temperature 21 ° C., photoperiod 16 hours / 8 hours and relative humidity 84%), a slow-acting fertilizer (Osmocote) was used. Transfer the seedlings to a 12 cm diameter pot filled with the same soil fertilized with g / l (soil), and then transfer to a greenhouse (type S2) which is watered twice a day for 2 minutes and controlled at 18 ° C. .

When flowers appear, place them in a bag (Crispac, reference number SM 570y 300 mm * 700 mm) in a way that prevents catabolism.

When the pod reaches maturity, the pod is harvested, dried and then ground. The seeds obtained are used for biochemical activity analysis. Transgenic offspring are selected by germination on a medium containing 100 to 150 mg / l kanamycin sulfate (depending on genotype). The operating conditions are the same as described above, except that germination is carried out in glass tubes with one seed per tube. Only plants with secondary roots are acclimated in the plant growth control room during the first three weeks before transfer to the greenhouse.

VI. Production Example of Transgenic Eggplant.

a) preparation of transgenic tobacco

For use in transformation experiments in Murashige and Cook's basal medium (1962) to which vitamins (Sigma Ref. No. M0404) according to Gamborg et al. (1968), 20 g / l sucrose and 8 g / l agar (Merck) were added. Tobacco (Nicotiana tabacum strains Xanthi NC and PBD6) are cultured in a laboratory. Before sterilization at 120 ° C. for 20 minutes, the pH of the medium is adjusted to 5.8 with caustic potassium solution. Every 30 days, the tobacco seedlings cut from the liver are transferred to the growth medium MS20.

All laboratory cultures are carried out in climatically controlled chambers under the conditions defined as follows:

Luminous intensity 30 μE.m ⁻² .S ⁻¹ ; Photoperiod 16 hours;

A temperature cycle of 26 ° C. during the day and 24 ° C. at night.

The transformation technique used was derived from Horsch et al. (1985).

Agrobacterium tumefaciens LBA4404, comprising pBIOC 29 or pBIOC 26 or pBIOC25 plasmids, was preliminary at 28 ° C. for 48 hours with stirring in LB medium (Sambrook et al., 1989) to which appropriate antibiotics (rifampicin and tetracycline) were added. Incubate. The preculture is then diluted 50-fold in the same medium and incubated under the same conditions. After overnight, the culture is centrifuged (3000 g, 10 minutes) and the bacteria are suspended 10-fold by suspending the same amount of liquid medium MS30 (30 g / l sucrose).

Explants of about 1 cm ² are cut from the seedling leaves described above. The explants are then contacted with the bacterial suspension for 1 hour, then quickly dried on filter paper and placed in a coculture medium (solid MS30).

After 2 days, the selection agent kanamycin (200 mg / l), bacteriostat ogmentin (400 mg / l), and the hormones necessary for induction of shoots (1 mg / l BAP, and 0.1 mg / l ANA) Seedlings are transferred to a Petri dish of regeneration medium MS30 containing). After two weeks of growth, seedlings are planted in the same medium. After an additional two weeks, the shoots are transferred to Petri dishes with developmental medium, consisting of MS20 medium with kanamycin and augmentin added. After 15 days, half of the shoots are transferred and planted. It takes about 20 days to take root, and at the end, the seedlings can be cut from the internodes and cloned or sprouted in the greenhouse.

b) preparation of transgenic tomatoes.

Tomato Seed cv. UC82B is sterilized for 15 minutes with 10% domestos and rinsed three times with sterile water. The final rinse is carried out for 10 minutes with stirring.

The above sterilized seeds were basted in Murashige and Cook's medium supplemented with vitamins (Thomas and Pratt, 1981) according to Nitsch, 30 g / l saccharose, and 8 g / l of Merck with a pH of 5.9 (1962). , 7 days in a climatically controlled chamber (luminance 30 μE. M ⁻² , s ⁻¹ , photoperiod 16 hours / 8 hours, 26 ° C.) in MSSV / 2 medium, Sigma Ref. M6899 / 2) or Germinate for 8 days.

The transformation technique used is derived from Fillatti et al. (1987).

Agrobacterium tumefaciens strain LBA4404, comprising pBIOC 25 or pBIOC 26 plasmids, is preincubated for 24 hours at 28 ° C. with stirring in LB medium with the appropriate antibiotics (rifampicin and tetracycline). The preculture is then diluted 50-fold with the same medium and incubated overnight under the same conditions. OD is measured at 600 nm, Agrobacteria are centrifuged (3000 g, 10 minutes) and suspended in KCMS liquid medium (described in the publication of Fillatti et al., 1987) to obtain OD 0.8 at 600 nm.

The improved technique was used at several stages of the protocol described by Fillatti et al. In 1987.

Except for supplementation with acetosyringone (200 mM) in KCMS medium, preculture and coculture of seedlings were performed as described by Fillatti et al. In 1987.

The 2Z washing medium differs in that 500 mg / l of Cytaxime was added instead of carbenicillin. The developmental medium used was Murashige and Cook added with vitamins according to Nitsch, 20 g / l saccharose, 50 mg / l kanamycin, 200 mg / l augmentin, 1 mg / l ANA and 0.5 mg / l zeatine Basal medium (Sigma MS6899).

VII. Preparation of Transgenic Corn

a) Preparation and use of maize callus as a target for gene transformation.

Whatever method used (electroporation, Agrobacterium, microfibers, particulate gun), genetically transforming corn usually requires the use of rapidly dividing undifferentiated cells with the ability to regenerate complete plants. One such type of cell is the fragile corn pear callus (called type II).

Callus is obtained from immature embryos of the H1 II genotype or from (A188 x B73) according to the method and medium described by Amstrong (Malze Handbook; (1994) M. Freeling, V. Walbot Eds .; pp. 665-671). . Callus obtained in this manner is propagated and maintained by successive transplantation into starting medium every 15 days.

Seedlings are then regenerated from the callus by altering the hormone and osmotic balance of the cells according to the methods described by Vain et al. (Plant Cell Tissue and Organ Culture (1989), 18: 143-151). The plant is then allowed to acclimate in a greenhouse to allow for mating or self-pollination.

b) Use of particulate guns for genetic transformation of corn

In this paragraph, the preparation and regeneration of the cell line required for transformation is described, and herein a gene transformation method for stably inserting the altered gene into the genome of a plant is described. The method relies on the use of the same particulate gun as described by J. Finer (Plant Cell Report (1992) 11: 323-328), wherein the target cell is the callus fragment described in paragraph 1. The fragments having a surface area of 10 to 20 mm ² are aligned at the ratio of 16 fragments per dish in the center of the Petri dish containing the same culture medium as the starting medium to which 0.2 M of mannitol + 0.2 M of sorbitol was added 4 hours before shelling. I was. The plasmid with the gene to be introduced is purified on a Qiagen® column according to the manufacturer's instructions. The plasmid is then precipitated on tungsten fine particles (M10) according to the method described by Klein (Nature (1987) 327: 70-73). Such coated particles are then fired towards the target cells using a gun according to the method described by J. Finer (Plant Cell Report (1992) 11: 323-328).

The callus dishes so screened are then sealed using Celloprice® and then incubated at 27 ° C. in the dark. The first planting is carried out after 24 hours, then every 15 days for 3 months, on the same medium as the starting medium to which the selection agent which can vary in form and concentration depending on the gene used (paragraph 3). Generally available screening agents consist of the active compound of a particular herbicide (Basta®, Round up®) or a specific antibiotic (hygromycin, kanamycin, etc.).

Three months later or sometimes earlier, a callus is obtained in which growth is not inhibited by the selection agent, usually and mostly consisting of cells derived from the division of cells into which at least one copy of the selection gene has been inserted into the gene pool. The frequency at which such callus is obtained is about 0.8 callus per hit dish.

The callus is identified, individualized, amplified and then incubated to regenerate the seedlings (see paragraph a). All of the above operations are carried out on a culture medium containing a selection agent to avoid any interference by untransformed cells.

Such regenerated plants are allowed to acclimate and then grown in greenhouses to allow for mating or self-pollination.

VIII. Analysis of the Expression of Dog Gastrointestinal Lipase in Transgenic Tobacco

a) protocol

The protocol for lipase extraction from tobacco leaves collected from plants in a greenhouse is as follows.

1 g of leaves (raw weight) was ground in liquid nitrogen and then 25 mM Tris HCl buffer (buffer A) or 1 mM EDTA, 10 mM β-mercapto at pH 7.5 with 1 mM EDTA and 10 mM mercaptoethanol. 5 ml of 25 mM glycine-HCl buffer (buffer B) at pH 3 with ethanol, 0.2% Triton X-100 and 250 mM NaCl was placed at 4 ° C. Total ground material was immediately centrifuged at 10,000 g for 15 minutes at 4 ° C.

For tobacco seeds, extraction was carried out in Buffer B in an amount of 0.1 g of seeds per 4 ml of buffer.

Lipase activity was measured using pH-STAT by titration by Gargouri et al. (1986) using tributyrin as substrate. An emulsion of tributyrin (4 ml per 30 ml emulsion) was prepared in the presence of bile salt (1.04 g / l), bovine albumin (0.1 g / l) and NaCl (9 g / l). The analysis included neutralization with a sodium carbonate solution of butyric acid liberated under the action of lipase at pH adjusted to 5.5 and 37 ° C. One unit of lipase corresponded to the amount of enzyme that caused the release of 1 micromole fatty acid for 1 minute at 37 ° C. at an optimal pH (5.5). Natural (purified) gastrointestinal lipases have specific activity against 570 units / mg of protein. Lipase activity can be measured in total ground material, precipitate or centrifuged supernatant.

Total soluble protein analysis was performed on the supernatant (buffer A) centrifuged by the method of Bradford (1976).

In addition, a sandwich ELISA test (Carriere et al., 1993) was performed on centrifuged supernatants using two groups of natural anti-DGL polyclonal antibodies. The first group includes antibodies that react with human gastric lipase and are purified by affinity using total antiserum against human gastrointestinal lipase grafted on a column of Affigel 10 (Aoubala et al., 1993). These antibodies were used to coat wells of Elisa plates at a concentration of 1 μl / ml. The second group includes antibodies that do not recognize human lipase. These antibodies bound to Protein A and then purified sequentially on Sepharose columns bound to biotin. Immobilization of the antibody was detected by indirect means of streptavidin / peroxidase conjugates in which enzymatic activity was demonstrated using the substrate o-phenylenediamine. The results produced are qualitative and recorded using the + and-symbols.

Extraction yield could be improved by adding CHAPS type detergent (3- (3-colamidopropyl) dimethyl-ammonio-1-propane sulfonate) (SIGMA) to the extraction buffer. In this case, the extraction yield in the supernatant actually increased to about 100% (see Table 1).

Lipase activity (U / gFW) in extracts generated from four tobaccos derived from genetic transformation with pBIOC25 in the presence of 1% CHAPS. plant NaCl 0.2 M / EDTA 1 mM pH 3.0 NaCl 0.2 M / EDTA 1 mM pH 3.0 + 1% CHAPS One 80 112 2 40 104 3 43 109 4 54 92

b) expression with plant signal peptide; analysis of tobacco leaves and seeds transformed with pBIOC25 and pBIOC26; Elisha test.

The results obtained in 98 T0 plants (15-20 leaf stages) of genotype Xanthi are shown in Table 2. All assays were performed in the ground mixture of leaves or seeds before centrifugation. For each construct, the measured activity shows a wide variety depending on the transformants. About 20% of the plants were inactive or too weak to detect activity. Average activity and maximum activity are shown in Table 2.

Total protein amounts were similar for the three constructs, with FW of leaves 7 and 8 mg / g and seed FW of 34, 31 and 38 mg / g.

Lipase mean activity in leaves transformed with construct pBIOC26 was 34 U / g (FW), 0.8% expression on total protein. In seeds, the average activity was 36 U / g (FW), which can be seen as 0.2%. The maximum activity produced in one of the transformants was 146 U / g (FW) (leaf; 2.5% expression) and 148 U / FWg (seed; 0.7% expression).

The enzymatic activity analyzed in plants transformed with construct pBIOC25 was similar. The mean activity in leaves was 34 U / g (FW) (0.8% expression) and maximum activity was 134 U / g (FW) (3% of total protein). In seeds, the average activity was 42 U / g (FW) and the maximum activity was 159 U / g (FW) (1%).

No activity was detected in the leaves of plants transformed with the construct pBIOC29 (seed specificity promoter). In seed, average activity was 12 U / g (FW) (0.1% expression) and maximum activity was 137 U / g (FW) (0.7).

Expression in leaves and seeds of the same transformation was generally appropriately related.

In addition, the results of the ELISA test were the same as the results of the enzyme activity, indicating that plants with lipase activity are positive results in the ELISA test.

The results obtained on these same plants then indicate that lipase activity may vary during the differentiation of the plant. In fact, plants with 3% expression on the total protein at the 12-15 leaf stage show 6% expression during subsequent analyzes (older flowering plants).

Expression of lipase in the leaves and seeds of xanthi tobacco. Structure organism Total protein Lipase activity Expression (% of total protein) mg / g (FW) U / g (FW) Min-max (average) Min-max (average) Min-max (average) pBIOC26 Leaf (n = 34) 3-15 (7) 0-145.6 (34.4) 0-2.5 (0.8) Seeds (n = 34) 24-43 (34) 0-147.6 (36.3) 0-0.7 (0.2) pBIOC25 Leaf (n = 35) 3-18 (8) 0-133.5 (34) 0-3 (0.8) Seeds (n = 35) 17-35 (31) 0-158.5 (41.9) 0-1 (0.3) pBIOC29 Seeds (n = 29) 29-55 (38) 0-137.4 (11.8) 0-0.7 (0.1) FW; Minimum fresh weight; Maximum value obtained with minimal expressing transformation; Value n obtained with the maximum expressing transformation; Number of transformations analyzed

b) expression with a signal peptide of RGL; Assay for lipase activity in tobacco leaves.

The results obtained from 44 T0 plants of genotype Xanthi and pBD6 (15-20 leaf stages) are shown below.

All analyzes were performed with a pulverized leaf mixture prior to centrifugation. The activity analyzed shows a wide variety depending on the transformants. About 20% of the plants showed no activity or were too weak to detect activity. Lipase mean activity in leaves transformed with construct pBIOC41 was 38 U per g FW for genotype Xanthi and 48 U per g FW for genotype PBD6. Maximum activity was 152 and 226 U per gram of FW, respectively.

IX. Analysis of the expression of dog gastrointestinal lipase in transgenic tomatoes.

a) protocol

The protocol for the extraction of lipase from tomato leaves and fruits was similar to that described for tobacco leaves, except 1 g of raw material was taken in 4 ml of Buffer B. Lipase activity was measured as described for tobacco leaves.

b) analysis on tomato fruit

The fruits of 30 primary transformants were analyzed.

The activity of the lipase analyzed in the fruit varied according to the fruit for the same transformant. Independently for the constructs tested (pBIOC25 or pBIOC26), it was 5 U per gram of FW for ripe fruit and 35 U per gram of FW for less ripe fruit.

It should be noted that activity decreased during fruit ripening. For example, for a given primary transformant, the lipase activity is 132 U per gram of green fruit FW 10 mm in diameter, 44 U per gram of red green fruit FW 33 mm in diameter and per gram of red fruit FW 45 mm in diameter. 36 U.

X. Immune Detection Against Western Types of Recombinant DGL.

a) DGL expressed in transgenic tobacco and rape leaves and seeds.

a.1) expression with plant signal peptide; Immune detection in tobacco and rape leaves and seeds transformed with pBIOC25, pBIOC26 and pBIOC29.

Western blots (Renart and Sandoval, 1984) immunodetection experiments on dog gastrointestinal lipases were carried out on rapeseeds extracted with tobacco leaves and tobacco proteins and buffers A and B (see extraction protocol above). To perform these experiments, the extracted protein (30 μg total protein per sample) was first added to Laem U.K. It was separated on a 12.5% modified polyacrylamide gel according to the technique of (1970) and then transferred onto a nitrocellulose membrane. Anti-dog lipase polyclonal antibodies generated in guinea pigs were detected using probes and anti-IgG antibodies of guinea pigs labeled with alkaline phosphatase.

A control protein that migrates in the form of a single band at an apparent molecular weight of about 50 kDa is natural dog gastrointestinal lipase (LC. FIG. 6). Migration of dog gastrointestinal lipase was slightly delayed in the presence of extracts of untransformed tobacco leaves (FIG. 6, LC + T).

No bands were detected in the protein extracts of untransformed tobacco and rape leaves and seeds. Lipase produced in tobacco leaves formed two bands. The quantitatively largest band had an apparent molecular weight of about 37 kDa which corresponded to the polypeptide mentioned above (Δ54). The smaller band had an apparent molecular weight of about 49 kDa which corresponded to the polypeptide (Δ4) mentioned above (FIG. 6, F). In the protein extracts of the seeds, only smaller molecular weight bands were observed visually (Figure 6, GT and GC).

a.2) expression with a signal peptide of RGL; Immune detection in tobacco leaves and seeds transformed with pBIOC41.

Western blots (Renart and Sandoval, 1984) were performed on the protein of tobacco leaves and seeds extracted with Buffer B (see above extraction protocol). The extracted protein was first separated on modified polyacrylamide gel (SDS-PAGE) according to the technique of Laemry (1970) and then transferred onto nitrocellulose membrane. Anti-dog lipase polyclonal antibodies generated in guinea pigs were detected using probes and anti-guinea pig antibodies labeled with alkaline phosphatase.

An example of a Western blot for tobacco leaves is shown in FIG. 7. A control protein that shifts to the formation of a single band at an apparent molecular weight of about 50 kDa is dog gastrointestinal lipase. No band was detected in the protein extract of the untransformed leaves of tobacco (T). Lipase produced in the leaf extract formed a main band at an apparent molecular weight of about 48-49 kDa, which was consistent with the polypeptide (D4) mentioned above.

In tobacco seeds transformed with the construct pBIOC41, the recombinant lipase is in the same form as in the leaves.

b) DGL: Deglycosylation experiments of protein extracts of transformed tobacco leaves.

The protocol for the extraction of proteins for deglycosylation experiments is as follows. 0.5 g of leaves (raw weight) were ground in liquid nitrogen and then in 4 ml of denatured buffer (1 mM β-mercaptoethanol, 25 mM EDTA and 1% SDS, 100 mM phosphate buffer, pH 7.5). It was set at ℃. The ground material was centrifuged at 10,000 g for 15 minutes at 4 ° C. The supernatant was incubated at 100 ° C. for 5 minutes to denature the protein and then centrifuged at 10,000 g for 2 minutes. The supernatant was then diluted 10-fold in deglycosylation buffer (100 mM phosphate buffer, pH 7.5) with 1% β-mercaptoethanol, 25 mM EDTA, 0.1% SDS and 1% octyl glucoside. Enzyme (N-glycosidase F, PNGase Boehringer) was added in an amount of 1 U per 100 μl of supernatant. An enzyme free control was used for each sample. Deglycosylation of control protein (dog or murine gastric lipase) occurred under the same conditions. Various protein samples were incubated for 8 hours at room temperature. Proteins were then separated by electrophoresis on polyacrylamide gels and transferred onto nitrocellulose membranes as described in the preceding paragraph.

According to the Western blot results, gastrointestinal lipase used as a control had an apparent molecular weight of about 50 kDa. After deglycosylation, its apparent molecular weight was only about 43 kDa.

After incubation without PNGase under the conditions described above, the lipase produced in tobacco leaves was found to form three bands of apparent molecular weight of 49 (polypeptide (Δ4)), 37 (polypeptide (Δ54)) and 28 kDa. . The band of molecular weight 28 kDa must be the result of proteolysis that occurs during 8 hours of incubation at room temperature. After deglycosylation, the molecular weight of the three bands, indicating that the protein produced in tobacco leaves was glycosylated, decreased to about 1-2 kDa.

c) DGL expressed in transgenic tomato leaves and fruits.

Western-type immunodetection experiments on dog gastrointestinal lipases were performed on protein of tomato leaves and fruits extracted with Buffer B (see above extraction protocol). To perform these experiments, the extracted proteins (15 μg and 6 μg total soluble protein for leaves and fruits, respectively) were separated on modified polyacrylamide gels as described in paragraph X.a).

The control protein, which migrates in the form of a single band at an apparent molecular weight of about 50 kDa, is a natural gastrointestinal lipase (Track 4, FIG. 8).

No band was detected in the protein extracts of undenatured tomato leaves and fruits.

Regardless of the construct used (pBIOC25 or pBIOC26), the lipase produced in tomato leaves and fruits was in the form of two bands. The largest quantitative band had an apparent molecular weight of about 37 kDa, which corresponds to the polypeptide mentioned above (Δ54). The smaller band had an apparent molecular weight of about 49 kDa, which corresponds to the polypeptide (Δ4) mentioned above (FIG. 8).

XI. Purification of Dog Gastrointestinal Lipase from Plants.

a) Purification of DGL from tobacco leaves.

Tributyrin (1 ml of tributyrin as a substrate at a temperature of 37 ° C. and pH adjusted to maintain the activity of dog gastrointestinal lipase produced in tobacco leaves at pH-status (Mettler-Toledo-DL25) Was emulsified in 29 ml of 0.15 M aqueous NaCl solution at Vortex). While the emulsion was maintained by vigorous mechanical agitation, the analysis involved neutralizing free butyric acid under the action of lipase by adding 0.02 N sodium carbonate. One lipase unit corresponded to one micromole of fatty acid free per minute under these pH and temperature conditions.

After the first chromatographic step, the lipase activity was determined by 0.5 ml of assay sample in an emulsion of 1 ml of tributyrin in 29 ml of a solution of 0.15 M NaCl, 2 mM sodium taurodeoxycholate and 1.5 μM of bovine serum albumin. It was explained according to the analysis (1986) described by Gargouri et al.

1 g of lyophilized leaves were ground in 30 ml of 20 mM glycine buffer, pH 2.5, 4 ° C., and the mixture was gently stirred for 15 minutes. While soaking in buffer, 1 N HCl was added to maintain the pH at 2.5. The product soaked in buffer was centrifuged at 15,000 g for 5 minutes. The pH of the supernatant was adjusted to 4 by addition of 1 N NaOH. After filtration with MIRACLOTH (Calbiochem), all supernatants were equilibrated in 10 ml (1.6 cm diameter) cation exchange resin in a buffer of 20 mM sodium acetate and 20 mM NaCl at pH 4 at a flow rate of 1 ml per minute. Used for column (S-Sepharose Fast Flow resin). 2 ml fractions were collected. After passage of the supernatant, the column was washed with 40 ml of equilibration buffer. Protein retained on the column was eluted according to the protocol below.

In a test conducted with 1 ml of analytical sample, linear in 20 mM sodium acetate buffer, pH 4.0, from 20 mM to 210 mM NaCl over 30 minutes, to elute the first peak of the protein containing no lipase activity. gradient,

Steady state in 210 mM NaCl for 20 minutes,

Linear gradient in 20 mM sodium acetate buffer at pH 4.0 of 210 mM to 500 mM NaCl over 30 minutes to elute the peak of Article 2. Lipase activity measured in 0.5 ml of assay sample eluted during this second gradient at an ionic strength of 300 to 400 mM.

The active fractions were collected and concentrated using an OMEGACELL concentrated cell (Filtron Technology Corporation) with a molecular weight limit of 30 kDa.

The pyruxate was dialyzed against 10 mM Tris-HCl buffer at pH 8 for 12 hours and then anion exchange resin (monoQ HR 5 / 0.5 cm in diameter and 5 cm in height) equilibrated in 10 mM Tris-HCl buffer at pH 8. 5-pharmaceutical). Lipase activity was measured in 0.5 ml of assay sample. The flow rate is maintained at 1 ml per minute, which is a pressure of 2.0 mPa. After eluting the unretained fractions and washing the column with 10 ml of 10 mM Tris-HCl buffer at pH 8, a linear gradient of ionic strength of 0 to 400 mM NaCl was applied over 60 minutes. Lipase activity was eluted at ionic strength between 100 mM and 200 mM.

The active fractions were collected and concentrated using an omegacell enrichment cell with a molecular weight limit of 30 kDa. The concentrate consisted of a purified form of dog gastrointestinal lipase extracted from tobacco leaves.

Concentration of the protein in the concentrated supernatant M. O. H. for the solution following the method of M. Bradford (1976) and after the first ion exchange chromatography. It was determined according to the method of Laurie (1951).

The various stages of purification are U.K. Analysis was performed by electrophoresis on modified polyacrylamide gel (SDS-PAGE 12.5%) according to the technique of Laemry (1970). On the one hand, proteins isolated in this way on polyacrylamide gels are detected by staining with Coomassie blue and on the other hand membranes in a buffer with 20 mM tris base, 150 mM glycine and 20% ethanol Transitions were made on nitrocellulose membranes by the technique of transblot SD, BIORAD, 2.3 mA per cm ² . Recombinant dog gastrointestinal lipase transferred on nitrocellulose membrane was detected by immunodetection according to the following protocol.

The desired protein for 1 hour at room temperature with dog gastrointestinal anti-lipase polyclonal antibodies produced in guinea pigs diluted 1 / 5,000 with PBS buffer, in which 5% skim milk powder and Tween 20 were added in an amount of 0.1%. To cover the

Wash the membrane for 10 minutes for each bath in three consecutive baths of PBS buffer, with 1% skim milk powder and Tween 20 added in an amount of 0.1%,

Labeled with anti-guinea pig antibody bound to peroxidase (Sigma) diluted 1 / 2,000 in PBS buffer, with 1% skim milk powder and 0.1% of Tween 20, at room temperature for 1 hour,

Wash the membrane for 10 minutes for each bath in three consecutive baths of PBS buffer, with 1% skim milk powder and Tween 20 added in an amount of 0.1%,

Detection by the action of peroxidase on 4-chloro-1-naphthol (Sigma) in the presence of H ₂ O ₂ which produces stable blue coloration over time.

Lipase produced in the leaves formed two bands of about 49 (polypeptide (Δ4)) kDa and 37 (polypeptide (Δ54)) kDa.

b) variants for purification of DGL from tobacco leaves.

Activity against dog gastrointestinal lipase produced in tobacco leaves was measured by the titration method described by Gargori et al. (1986) using pH-stat (METTLER-TOLEDO-DL25).

Analysis for short chain triglycerides was carried out using tributyrin as substrate. 1 ml of tributyrin was emulsified in 29 ml of an aqueous solution of 0.15 M NaCl, 0.5 μM bovine serum albumin (BSA) and 2 mM sodium taurodeoxycholate (NaTDC). The adjusted pH was maintained at 5.0 and the temperature at 37 ° C.

While maintaining the emulsion with vigorous mechanical stirring, the analysis involved neutralizing the free butyric acid by adding 0.02 N sodium carbonate under the action of lipase.

Assay for long chain triglycerides using water emulsion using 30% refined soybean oil, 1.2% refined egg phospholipid, 1.67% anhydrous glycerol (purchased from PHRMACIA AB, Intralipid ^™ 30%-Stockholm, Sweden) It was carried out by. 10 ml of this suspension were emulsified in 20 ml of an aqueous solution of 0.15 M NaCl, 30 μM BSA and 3.5 mM CaCl ₂ . The adjusted pH was maintained at 4.0 and the temperature at 37 ° C.

After maintaining the emulsion with vigorous mechanical stirring, while jumping the pH from 4 to 9, the analysis consisted of neutralizing free fatty acids by the addition of 0.2 N sodium carbonate under the action of lipase.

One unit lipase corresponded to one micromole of fatty acid liberated per minute under the conditions defined for each pH and temperature.

2 g of lyophilized leaves were ground in 60 ml of 0.2 M NaCl, pH 3 at 4 ° C. and the mixture was gently stirred at 4 ° C. for 15 minutes. While soaked in buffer, 1 N HCl was added to maintain pH at 3. The product (homogenate) soaked in buffer was centrifuged at 10,000 g for 10 minutes. After filtration with MIRACLOTH and 0.45 μ Millipore filters, all supernatants were buffered at pH 3 with 20 mM sodium acetate and 0.2 M NaCl at a flow rate of 8 ml / min (240 cm h ⁻¹ ). Was injected into an equilibrated cation exchange resin column (RESOURCE S 6 ml- weakly-16 mm id × 30 mm).

After passage of the unretained fractions, the column was washed 30 times using equilibration buffer as the volume of the column. The protein retained on the column was eluted with a linear gradient in 20 mM sodium acetate buffer, pH 3 of 0.2 to 0.5 M NaCl, in 7 column volumes. 4 ml fractions were collected.

Lipase activity measured on 0.5 ml of assay sample eluted at an ionic strength of 0.35 M NaCl.

The active fractions were collected and concentrated using an omegacell enrichment cell (Filtron Technology Corporation) with a molecular weight limit of 30 kDa.

The measurement for protein concentration is OH. It was carried out in accordance with the Law of Law (1951).

Examples of polyacrylamide gels on nitrocellulose membranes and the results of the transfer of these gels and detection from dog gastrointestinal anti-lipase antibodies were shown (FIGS. 9 and 10) and activators (ECL Western blotting-AMERSHAM LIFE SCIENCE) Detection was carried out by the action of peroxidase on Luminol in the presence of and recorded on photographic film.

The presence of glycan residues in the protein was determined according to the following protocol:

Immobilization of Proteins on Microcellulose Membranes

Treated with periodate

Specific reaction with streptavidin combined with alkaline phosphatase

Coloring reaction with alkali phosphate (GLYCOTRACK OXFORD GLYLOSYSTEM).

Examples of membrane detection of glycan residues are shown (Table 11).

High performance liquid chromatography was used to ensure the purity of the fractions.

Chromatograph Waters 625 LC

Diode Alignment Detector Waters 991 (WATERS 991)

Column bidak C4 (VYDAC C4)

Elution condition:

Time (min) Flow rate (ml / min) % A % B 0 One 100 0 35 One 40 60 40 One 40 60 45 One 20 80 50 One 100 0 Buffer A: 89.9% H ₂ O, 10% acetonitrile and 0.1% trifluoroacetic acid buffer B: 100% acetonitrile

Purification Table : Recombinant DGL

Total Unit * Mg of protein Inactivity Refinement factor yield Homogenate 3200 Of Of Of Of Supernatant 2800 230 13 One 88% Emissions S 1600 6 250 20 50% Tributyrin Unit

Comparative specific activity against natural dog gastrointestinal lipase (n-DGL) and recombinant dog gastrointestinal lipase (r-DGL)

n-DGL * R-DGL Extract from Tobacco Leaves Tributyrin 570 U / mg 250 U / mg Intra Lipid 30% 1000 U / mg 950 U / mg * Carriere et al (1991) Eur. J. Biochem, 202 , 75-83.

c) purification of DGL from rapeseed

Activity against recombinant DGL produced in rapeseed seeds was measured in the same manner as when extracted from leaves.

10 g of rapeseed was ground in liquid nitrogen. The resulting powder was degreased with hexane as first soak at 4 ° C. with gentle stirring for 12 hours. The whole amount was decanted and hexane was removed. The powder was rinsed twice with 100 ml of hexane with gentle stirring at 4 ° C. for 1/2 hour for each rinse. The hexanes were decanted off. The powder was dried on a rotary evaporator (HEIDOLPH 94200). The degreased powder was stored at -20 ° C.

Extraction of DGL was performed from powder degreased by immersion at 4 ° C. in an aqueous solution of 0.2 M NaCl, pH 3 (1 N HCl), at a rate of 2 ml of aqueous solution per 0.1 g of powder. The product by dipping was centrifuged at 10,000 g at 4 ° C. for 10 minutes.

The pellet was removed. The supernatant constituted the seed extract.

Seed extracts were dialyzed against a buffer of pH 4 with 10 mM sodium acetate, 140 mM NaCl, 3 mM KCl, and then produced from guinea pigs bound to resin (hydrazide avidgel-BIOPROBE INTERNATIONAL, Inc.). It was applied to an immunoaffinity column consisting of dog gastrointestinal anti-lipase polyclonal antibodies.

Resin / seed extract contact was carried out for 30 minutes with gentle stirring at 4 ° C. The resin was then rinsed with 10 column volumes of 10 mM sodium acetate, 150 mM NaCl, 3 mM KCl, pH 4 buffer. DGL was extracted with 5 column volumes of 0.2 M glycine, 150 mM NaCl, pH 2.8 buffer. The collected fractions contained 1 / 20th V / V of 1 M Tris buffer, pH 9, with a volume equal to 1 column volume. Analysis by electrophoresis on polyacrylamide gel in denaturing medium (see FIG. 12) shows that the molecular weight of the protein is about 37 kDa.

XII. Synthesis of Fatty Acid Esters.

-In the form of an enzyme preparation either fixed (lipozyme (NOVO)) or not fixed (rat gastrointestinal lipase (JO 4002)),

A test for lipase supplied in the form of tomato seeds transformed with the gene of dog gastrointestinal lipase (tobacco T14-44 0.85% expression) was performed with untransformed rapeseed.

The esterification reaction was carried out at 37 ° C. for 16 hours in a glass bottle of sealed stopper located on a stir bench (250 rpm). Hexane was used as the organic solvent for dissolving fatty acids. Methanol was added stoichiometrically about the theoretical amount of triacylglycerol contained in rapeseed seeds.

The main component in the fatty acids of rape is oleic acid, and the selected comparative control is a methyl ester of oleic acid. Synthesis was observed by thin layer chromatography (TLC). The moving solvent was a mixture of hexane, diethyl ether and water (70: 10: 1). Ethanol containing sulfuric acid (5%) was sprayed and subjected to detection on a plate under high temperature conditions.

In the first test, 27 μl of methanol (0.66 mmol) and 0.02 g of lipozyme were added to 0.2 g (0.22 mmol) of rapeseed oil. No dot appeared at the level of the reference methyl oleate (FIG. 13, column 2).

In a second test, rapeseed oil was replaced with 0.5 g of rapeseed crushed in dry state and 1 ml of hexane was added. Methyl esters were synthesized (FIG. 13, column 5).

In the next test, the above conditions were repeated with the following modifications. The amount of lipozyme was reduced to 0.006 g and lipozyme was replaced with 0.007 g of rabbit gastrointestinal lipase (JO 4002). Methyl oleate was synthesized in the presence of lipozyme but not JO 4002 (FIG. 14, column 2).

Finally, in the final test, lipase was fed to transformed tobacco seeds (1 g of tobacco seeds). It was found to have the properties of methyl esters (FIG. 15, column 3).

In conclusion, the results demonstrate that when the rapeseed extract, alcohol and recombinant dog gastrointestinal lipase produced by transgenic tobacco are present together, an esterification reaction can be obtained which results in the synthesis of a methyl ester that can be used as biofuel. It was.

Description of the drawing:

1: nucleotide sequence of a cDNA encoding DGL,

2: amino acid sequence of DGL,

3: nucleotide sequence derived from cDNA encoding DGL, encoding the DGL shown in FIG. 2,

4: nucleotide sequence of cDNA encoding HGL and amino acid sequence of HGL,

5: amino acid sequence of HGL,

6: Immunodetection of recombinant polypeptides produced by xanthi tobacco leaves and seeds and rapeseed transformed with constructs pBIOC26, pBIOC25 and pBIOC29; E, molecular weight range; LC, dog gastric lipase; F, tobacco leaves and GT transformed with construct pBIOC25, tobacco seeds; GC, Rapeseed transformed with construct pBIOC29; T, nontransformed tobacco leaves,

7: Immune detection of recombinant polypeptides produced by tobacco leaves transformed with constructs pBIOC25 and pBIOC41; E, molecular weight range; LC, dog gastric lipase; 1 and 3, tobacco leaves transformed with construct pBIOC41; 2, tobacco leaves transformed with construct pBIOC25; T, nontransformed tobacco leaves,

8: Immunodetection of recombinant polypeptides produced by tomato leaves and fruits transformed with constructs pBIOC25 and pBIOC26:

T1: Untransformed Tomato Fruit

1 and 3: transformed tomato fruit

2: transformed tomato leaves

R: molecular weight range

LC: Dog Gastrointestinal Lipase

T2: Untransformed Tomato Leaf.

Figure 9: Analysis by electrophoresis on polyacrylamide gel in unmodified medium (SDS-PAGE).

1: Rapeseed Extract, 2: Recombinant Dog Gastrointestinal Lipase Produced from Tobacco Leaves

3: natural dog gastrointestinal lipase, 4: molecular weight range.

10: Immunological detection of the above-described assays after transition on nitrocellulose membrane

1: natural dog gastrointestinal lipase, 2: recombinant dog gastrointestinal lipase produced from tobacco leaves

3: Rapeseed Extract.

Figure 11: Detecting the presence of glycan residues from polyacrylamide gels after transition onto nitrocellulose membrane

1: Rapeseed Extract, 2: Recombinant Dog Gastrointestinal Lipase Produced from Tobacco Leaves

3: Natural Dog Gastrointestinal Lipase.

12: SDS-PAGE analysis of immunoassay of r-DGL produced in Rapeseed

1: natural dog gastrointestinal lipase, 2: recombinant dog gastrointestinal lipase

3 to 6: unretained fraction eluted from the immunopurification column.

13: detection on TLC plate; 1: rapeseed oil, enzyme free; 2: rapeseed oil + lipozyme; 3: methyl ester of oleic acid; 4: rapeseed, enzyme free; 5: rapeseed + lipozyme,

14: detection of TLC plates; 1 and 4: rapeseed without enzyme; 2: rapeseed + JO4002; 3: methyl ester of oleic acid; 5: rapeseed + lipozyme,

15: detection on TLC plate; 1: monoolein, diolein, triolein; 2: methyl ester of oleic acid; 3: rapeseed + transformed tobacco seeds.

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<Sequence table>

(1) General Information:

(i) Applicant:

(A) Name: Biochem S.A.

(B) Street: Avenue de Rende 24

(C) City: Oviere

(E) Country: France

(F) Zip code: 63170

(A) Name: Engistu de Lesser Juvenal

(B) street: Rue de la Rogue 3-9

(C) City: Fresne Cedex

(E) Country: France

(F) Zip Code: 94265 Sedex

(ii) Name of the Invention: Recombinant zodiac lipase and polypeptide derivatives produced by plants, methods of making and uses thereof

(iii) SEQ ID NO: 6

(iv) computer-readable form

(A) Media type: floppy disk

(B) Computer: IBM PC Compatible

(C) working system: PC-DOS / MS-DOS

(D) Software: Patterned Release # 1.0, Version # 1.25 (EPO)

(vi) Prior application data:

(A) Application number: FR 9504754

(B) Application date: April 20, 1995

(2) Information about SEQ ID NO: 1

(i) Sequence Features:

(A) Length: 1528 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: cDNA to mRNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..1137

(xi) Sequence description:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 379 amino acids

(B) Type: Amino Acid

(D) topology: linear

(ii) Molecular Form: Protein

(xi) Sequence description:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 1048 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: cDNA to mRNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..975

(xi) Sequence description:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 325 amino acids

(B) Type: Amino Acid

(D) topology: linear

(ii) Molecular Form: Protein

(xi) Sequence description:

(2) information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 1198 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) topology: linear

(ii) molecular form: cDNA to mRNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..1125

(xi) Sequence description:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 375 amino acids

(B) Type: Amino Acid

(D) topology: linear

(ii) Molecular Form: Protein

(xi) Sequence description:

Claims (33)

On the one hand any mammalian preduodenal lipase, ie, at least about 75%, in particular from about 77% to 85% homology with a nucleotide sequence of at least 70%, in particular from about 80% to about 90% CDNA, which encodes any mammalian total duodenal lipase having homology and having properties that are acid resistant and active at a pH of about 1 to about 5, especially about 1.5 to about 2, more specifically any mammalian stomach CDNA encoding a lipase, or any polypeptide derived from said duodenal lipase by addition and / or inhibition and / or substitution of one (or more) amino acid (s) having the above properties of pleural lipase CDNA, on the other hand, a component which allows plant cells to produce a twentieth lipase encoded by said cDNA or to produce an induced polypeptide as defined above. Mammalian recombinant transduodenal lipase, as defined above, in the form of an active enzyme from a transformed plant cell or a plant obtained from the cell, the recombinant nucleotide sequence comprising a transcriptional promoter and a transcription terminator recognized by the transcriptional machinery of the plant cell Use for transforming plant cells to obtain species (or more) polypeptide (s). The addition and / or inhibition and / or inhibition of cDNA, or one (or more) nucleotide (s) shown in FIG. 1, encoding on the one hand the dog gastrointestinal lipase (DGL) shown in FIG. 2. ) Addition and / or inhibition of a nucleotide sequence capable of encoding a polypeptide of the same amino acid sequence as the amino acid sequence of DGL shown in FIG. 2 derived from the cDNA by substitution, or one (or more) amino acid (s) And / or nucleotides encoding a polypeptide having lipase activity, derived from DGL by substitution, on the other hand a component such that the plant cell produces the cDNA or polypeptide encoded by the derived sequence, in particular plant cells Obtained from a transformed plant cell or a recombinant nucleotide sequence comprising a transcriptional promoter and transcriptional terminator recognized by a transcriptional apparatus of Use for transforming a plant cell from a plant in order to obtain an active enzyme form of the recombinant DGL or the above-defined one (or more) derived polypeptide (s). The addition and / or inhibition and / or inhibition of cDNA, or one (or more) nucleotide (s) shown in FIG. 2, encoding on the one hand the human gastrointestinal lipase (HGL) shown in FIG. 5. ) The addition of a nucleotide sequence capable of encoding a polypeptide having an amino acid sequence identical to the amino acid sequence of HGL shown in FIG. 5 derived from the cDNA by substitution, or one (or more) amino acid (s) and / or) Nucleotides encoding polypeptides having lipase activity, derived from HGL by inhibition and / or substitution, on the other hand components which allow the plant cells to produce polypeptides encoded by the cDNA or the derived sequences, in particular plants Transformed plant cells into or from recombinant nucleotide sequences comprising transcriptional promoters and transcriptional terminators recognized by the transcriptional machinery of the cell For transforming plant cells in order to obtain an active enzyme form of the recombinant HGL, or the definition of one (or more) derived polypeptide (s) from the obtained plant uses. On the one hand, the cDNA shown in FIG. 1 encoding the dog gastrointestinal lipase (DGL) shown in FIG. 2, or the addition and / or inhibition and / or substitution of one (or more) nucleotide (s) of the cDNA Derived from the addition and / or inhibition and / or substitution of a nucleotide sequence capable of encoding a polypeptide of the same amino acid sequence as the amino acid sequence of DGL shown in FIG. 2 or one (or more) amino acid (s) Nucleotides encoding polypeptides having lipase activity, derived from DGL, on the other hand by components that allow plant cells to produce the cDNA or polypeptides encoded by the derived sequences, in particular by transcriptional machinery of plant cells. A recombinant nucleotide sequence comprising a transcriptional promoter and a transcription terminator. On the one hand, the cDNA shown in FIG. 2 encoding human gastrointestinal lipase (HGL) shown in FIG. 5, or the addition and / or inhibition and / or substitution of one (or more) nucleotide (s) of the cDNA The addition and / or inhibition and / or substitution of a nucleotide sequence capable of encoding a polypeptide having an amino acid sequence identical to that of the HGL shown in FIG. 5, or one (or more) amino acid (s), derived from Nucleotides encoding polypeptides having lipase activity, derived from HGL, on the other hand by components which allow plant cells to produce the cDNA or polypeptides encoded by the derived sequences, in particular by transcriptional machinery of plant cells A recombinant nucleotide sequence comprising a recognized transcriptional promoter and transcriptional terminator. The method according to claim 4 or 5, Terminator polyA 35S of cauliflower mosaic virus (CaMV) or terminator polyA NOS of Agrobacterium tumefaciens, downstream of the cDNA or its derived sequence, -Upstream of the cDNA or its derived sequence, promoter 35S or double stranded promoter 35S (pd35S) of CaMV, or promoter pCRU of the Cruciferin gene of radish, or promoter pGEA1 or pGEA6 of Arabidopsis thaliana, or agrobacterium tumefaciens A recombinant nucleotide sequence comprising a chimeric-promoter pSP, or a rice promoter pAR-IAR, or a corn promoter pγzeine. The recombinant nucleotide sequence of claim 4, comprising a sequence encoding a peptide that directs the recombinant DGL and / or derived polypeptide (s) to specific organs of the plant cell. The method according to any one of claims 4, 6 and 7, A nucleotide sequence comprising a sequence encoding the promoter pd35S of CaMV, the signal peptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV in the 5 '→ 3' direction ( pd35S-PS-DGL), A nucleotide sequence comprising a sequence encoding the promoter pd35S of CaMV, the prepropeptide of sporamin A in the 5 '→ 3' direction, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV (called pd35S-PPS-DGL), The sequence encoding the promoter pd35S of CaMV, part of the signal peptide of RGL in the 5 '→ 3' direction (the first 19 nucleotides with 9 nucleotides encoding the last three C-terminal amino acids shown in the examples of the specification) A nucleotide sequence (called pd35S-RGLSP-DGL) which sequentially comprises the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 immediately after this sequence, A nucleotide sequence comprising a promoter pCRU of cruciferin in the 5 '→ 3' direction, a sequence encoding a signal peptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and a terminator polyA 35S of CaMV (called pCRU-PS-DGL), A nucleotide comprising the promoter pCRU of cruciferin in the 5 '→ 3' direction, the sequence encoding the prepropeptide of sporamin A, the nucleotide sequence shown in FIG. 3 immediately after this sequence, and the terminator polyA 35S of CaMV. Sequence (called pCRU-PPS-DGL), -A sequence encoding a part of the signal peptide of the cruciferin promoter pCRU, RGL in the 5 '→ 3' direction (same as above), and the terminator polyA 35S of the cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. Nucleotide sequence comprising in sequence (called pCRU-RGLSP-DGL), -A sequence encoding the part of the signal peptide of the Arabidopsis thaliana promoter pGEA1, RGL in the 5 '→ 3' direction (same as above), the terminator polyA 35S of the cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. A nucleotide sequence comprising in sequence (called pGEA1-RGLSP-DGL), -A sequence encoding a part of the signal peptide of RAVIDOPDIS THALIANA promoter pGEA6, RGL in the 5 '→ 3' direction (same as above), the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 or 3 immediately after this sequence A nucleotide sequence comprising in sequence (called pGEA6-RGLSP-DGL), A sequence encoding a part of the rice promoter pAR-IAR, a signal peptide of RGL in the 5 '→ 3' direction (same as above), and the terminator polyA of cDNA and CaMV shown in FIG. 1 or 3 immediately after the sequence. A nucleotide sequence comprising the terminator polyA NOS of 35S or Agrobacterium tumefaciens (called pAR-IAR-RGLSP-DGL), The sequence encoding the promoter pγzeine of corn, part of the signal peptide of RGL in the 5 '→ 3' direction (same as above), and the terminator polyA 35S of cDNA and CaMV shown in FIG. 1 or 3 immediately after this sequence. Nucleotide sequence comprising in sequence (called pγzeine-RGLSP-DGL), A sequence encoding the promoter pγzeine of corn and a part of the signal peptide of RGL in the 5 '→ 3' direction (same as above), the sequence encoding the cDNA and tetrapeptide KDEL shown in FIG. 1 or 3 immediately after the sequence. And a nucleotide sequence comprising the terminator polyA 35S of CaMV in order (referred to as pγzeine-RGLSP-DGL-KDEL). Recombinant nucleotide sequence, characterized in that selected from. The method according to any one of claims 5 to 7, A promoter pSP of Agrobacterium tumefaciens, a sequence encoding a signal peptide of HGL, a nucleotide sequence shown in FIG. 4 immediately after this sequence, and a terminator polyA 35S of CaMV in the 5 '→ 3' direction Nucleotide sequence (called pSP-HGLSP-HGL), A promoter pSP of Agrobacterium tumefaciens, a sequence encoding a signal peptide of HPL, a nucleotide sequence shown in FIG. 4 immediately after this sequence, and a terminator polyA 35S of CaMV in the 5 '→ 3' direction Nucleotide sequence (called pSP-HPLSP-HGL), The promoter pSP of Agrobacterium tumefaciens, part of the signal peptide of RGL (described in claim 8) in the 5 '→ 3' direction (described in claim 8), the nucleotide sequence shown in FIG. 4 immediately after this sequence and the CaMV Nucleotide sequence comprising the terminator polyA 35S in sequence (referred to as pSP-RGLSP-HGL) Recombinant nucleotide sequence, characterized in that selected from. A vector, in particular a plasmid, comprising a nucleotide sequence according to any one of claims 4, 6, 7, or 8 at a site not essential for its replication. A vector, in particular a plasmid, comprising a nucleotide sequence according to any one of claims 5, 6, 7, or 9 at a site not essential for its replication. A cell host transformed with a vector according to claim 10 or 11, in particular bacteria such as Agrobacterium tumefaciens. The recombinant sequence according to any one of claims 4, 6, 7, and 8, with the help of the cell host according to claim 12, which is itself transformed with the vector according to claim 10, is the genome of the plant cell. Transforming the plant cells in a manner integrated into the If appropriate, generating a transformed plant from said transformed cells, Lipase activity derived from said recombinant DGL by addition and / or inhibition and / or substitution of recombinant DGL and / or in particular one (or more) amino acid (s) produced in said cell or said transformed plant. Recovering the polypeptide (s) having a specific viability by extraction, and, where appropriate, by purification after extraction A method for producing a polypeptide (s) having lipase activity derived from recombinant DGL in the form of an active enzyme or recombinant DGL by the above method. The recombinant sequence according to any one of claims 5, 6, 7, and 9, with the help of the cell host according to claim 12, which is itself transformed with the vector according to claim 11 Transforming the plant cells in a manner integrated into the If appropriate, generating a transformed plant from said transformed cells, Lipase activity derived from said recombinant HGL by addition and / or inhibition and / or substitution of recombinant HGL and / or in particular one (or more) amino acid (s) produced in said cell or said transformed plant. Recovering the polypeptide (s) having a specific viability by extraction, and, where appropriate, by purification after extraction A method for producing a polypeptide (s) having lipase activity derived from recombinant HGL in the form of an active enzyme or recombinant HGL by the above method. One (or more) recombinant nucleotide (s) according to any one of claims 4 to 9, and suitably recombinant DGL and / or recombinant HGL and / or Genetically transformed rape, tobacco, corn, peas, tomatoes, carrots, wheat, barley, potatoes, soybeans, sunflowers with enzymatic activity, in particular lipase activity, , A plant selected from lettuce, rice, alfalfa and beet, or parts of a plant, in particular leaves and / or fruits and / or seeds and / or plant cells. Polypeptide (s) derived from said recombinant DGL by addition and / or inhibition and / or substitution of recombinant DGL and / or one (or more) amino acid (s) by chromatography performed on the obtained enzyme extract. Consisting of recombinant DGL having the enzymatic activity of the amino acid sequence shown in FIG. 2, or amino acids at positions 55 to 379 of FIG. 2, obtained in essentially pure form by carrying out the method of claim 13 comprising a purification step From the recombinant DGL by addition and / or inhibition and / or substitution of one (or more) amino acid (s), such as polypeptide (Δ54), polypeptide consisting of amino acids at positions 5 to 379 of Figure 2 (Δ4) Polypeptides with Induced Lipase Activity. Recombinant HGL by chromatography performed on the obtained enzyme extract and / or polypeptide (Δ54HGL) consisting of amino acids at positions 74 to 398 of FIG. 5, polypeptide (Δ4HGL) consisting of amino acids at positions 24 to 398 of FIG. 5 and Essentially by the practice of the method of claim 14 comprising the step of purifying the polypeptide (s) derived from said recombinant HGL by addition and / or inhibition and / or substitution of one (or more) amino acid (s). Recombinant HGL having enzymatic activity of the amino acid sequence shown in FIG. 5, obtained in pure form, or from said recombinant HGL by addition and / or inhibition and / or substitution of one (or more) amino acid (s) Polypeptides with Induced Lipase Activity. According to claim 13, characterized in that it comprises one (or more) derived polypeptide (s) such as recombinant DGL and / or polypeptide (Δ54) and / or polypeptide (Δ4) as defined in claim 16. Plant extract having enzymatic activity obtained by the practice of the method. According to claim 14, characterized in that it comprises one (or more) derived polypeptide (s) such as recombinant HGL and / or polypeptide (Δ54HGL) and / or polypeptide (Δ4HGL) as defined in claim 17. Plant extract having enzymatic activity obtained by the practice of the method. 20. The method of claim 18 or 19, wherein the weight percent of the recombinant polypeptide having enzymatic activity is about 0.1% to 20%, especially about 1% to 15%, based on the total weight of the protein present in the extract, about 0.5 U to about 1,000 U / g (raw weight of leaves (FM)), especially about 10 U / g (FW) to about 300 U / g (FW), or about 1 U to about 5,000 U / g (FM of seed ), In particular a plant extract having an enzymatic activity, characterized in that it corresponds to a measurement of from about 10 U / g (FW) to about 1,000 U / g (FW). Healthy subjects or individuals suffering from one or more lesions that may or may not affect the level of production of gastrointestinal and / or pancreatic lipases, especially those who are undergoing medical treatment that alter the mechanism of fat absorption. Medications for promoting the absorption of animal or vegetable fats digested by, in particular for the treatment of lesions associated with a lack of lipase (especially gastrointestinal and / or pancreatic lipases) in the individual, in particular cysts such as cystic fibrosis and pancreatic deficiency A genetically transformed plant or part of a plant, in particular leaves and / or fruits and / or seeds, or plant cells, or a recombinant DGL according to claim 16, for the manufacture of a medicament 19. A derived polypeptide, or a recombinant HGL according to claim 17 or a polypeptide derived therefrom, or according to any one of claims 18-20. The use of bovine extracts. Recombinant DGL and / or HGL and / or one (or more) derived polypeptide (s) according to claim 16 or 17, and / or one according to any one of claims 18 to 20 ( Or above) an enzyme extract, if appropriate, together with a pharmaceutically acceptable excipient. Food, especially functional foods for promoting the absorption of animal or vegetable fats digested by healthy individuals or individuals suffering from one or more lesions that may or may not affect the production level of the stomach and / or pancreatic lipase. A genetically transformed plant or part of a plant, in particular leaves and / or fruits and / or seeds, or plant cells, or a recombinant DGL according to claim 16 or derived therefrom for the preparation of Use of a purified polypeptide, or a recombinant HGL according to claim 17 or a polypeptide derived therefrom, or an enzyme extract according to any one of claims 18 to 20. One (or more) plant (s) according to claim 15 or part of this plant, and / or recombinant DGL according to claim 16 or 17 and / or recombinant HGL and / or 1 derived therefrom. A functional food comprising a species (or more) polypeptide (s) and / or one (or more) enzyme extract (s) according to any one of claims 18 to 20. Genetically transformed plant according to claim 15, or part of a plant, in particular leaves and / or, for carrying out enzymatic reactions in industrial, fertilizer or agricultural sectors, in particular in the local, provincial and dairy industries. Fruit and / or seed or plant cells or recombinant DGL according to claim 16 or a polypeptide derived therefrom, or recombinant HGL according to claim 17 or a polypeptide derived therefrom, or any of claims 18 to 20 Use of an enzyme extract according to. Genetically transformed plants or parts of plants, in particular leaves and / or fruits and / or seeds, according to claim 15 in industrial, fertilizer or agricultural-related industries, in particular in the local, local chemical and dairy industries, or By plant cells, or recombinant DGL or polypeptides derived therefrom, or recombinant HGL or polypeptides derived therefrom, or the enzyme extract according to any one of claims 18-20. A method of biomaterial conversion or biocatalysis by enzymes by performing one or more enzymatic reactions carried out. One or more enzyme extract (s) having enzymatic activity according to any one of claims 18 to 20 and / or recombinant DGL and / or recombinant HGL and / or according to claim 16 or 17. Enzyme preparation for industrial, fertilizer or agricultural related industrial use which comprises one (or more) polypeptide (s) derived therefrom and which can be used in the method according to claim 26. A genetically transformed plant or part of a plant, in particular leaves and, according to claim 15, for carrying out a biomaterial conversion reaction or biocatalysis by an enzyme, such as an enzyme hydrolysis or an ester transfer reaction on an industrial scale. Or) the use of fruit and / or seed or plant cells. A method of biocatalysis using the genetically transformed plant or part of a plant according to claim 15, in particular leaves and / or fruits and / or seeds or plant cells, used both as an enzyme source and as a reaction substrate. Use for the biofuel manufacture of the genetically transformed plant or plant according to claim 15, in particular leaves and / or fruits and / or seeds or plant cells. A process for producing a biofuel by adding alcohol, in particular methanol or ethanol, to the powder of all or part of the genetically transformed plant according to claim 15 and recovering the biofuel, in particular by filtration. Ester of plant fatty acids, in particular methyl ester of oleic acid, obtained by carrying out the process according to claim 31. A biofuel obtained by carrying out the process according to claim 31 comprising esters of plant fatty acids, in particular methyl esters of oleic acid.